Nuclear factor kappaB inducing factor

ABSTRACT

The present invention is directed to nuclear factor κB (NFκB)-inducing factor polypeptides (NFIF polypeptides) which are capable of inducing NFκB. The present invention includes within its scope NFIF polypeptides, including NFIF-14b and NFIF-7a, DNA, including cDNA, encoding these polypeptides, and expression vectors capable of expressing NFIF polypeptides. Also included are methods and compositions for increasing NFκB induction in a patient, methods and compositions for lowering NFκB induction in a patient, methods for inhibiting inflammation, and methods for manufacutre of a medicament intended for the treatment and/or prevention of an NFκB-regulated inflammatory response. In addition, methods for determining whether a test compound inhibits or enhances the activity of NFIF polypeptides are provided.

FIELD OF THE INVENTION

[0001] Nuclear factor κB (NFκB) comprises a family of eukaryotictranscription factors involved in regulation of genes involved in immuneresponses and other cellular functions. In some instances, NFκBactivation leads to an inflammatory response which ultimately results ina disease state. Accordingly, it would be desirable to develop means forcontrolling induction of NFκB. The present invention providespolypeptides which are involved in the induction of NFκB and which maybe used to enhance NFκB expression or activation or to identify andprepare inhibitors Of NFκB expression or activation.

REPORTED DEVELOPMENTS

[0002] Nuclear factor κB (NFκB) comprises a family of transcriptionfactors found in almost all eukaryotic cells. NFκB plays a role in theregulation of genes involved in tissue inflammation, cellularproliferation, and cellular differentiation.

[0003] A number of studies have examined the relationship betweendisease states and expression of the subunit proteins which compriseNFκB or activation of NFκB already present in a cell. Li et al. studiedthe regulation of NFκB by the HTLV-1 Tax protein demonstrating that theability of Tax to activate the NFκB pathway plays an essential role inHTLV-1-induced cellular transformation (Li et al., Gene Expr., 7, 4-6,233-245 (1999)). Lentsch and Ward studied the activation and regulationof NFκB during acute inflammation and described the relationship betweenNFκB and the inhibiting proteins of the IKappaB family (Lentsch et al.,Clin. Chem. Lab. Med., 37, 3, 205-208 (1999)). Visconti et al.investigated the role of NFκB in thyroid carcinogenesis by analyzingthyroid carcinoma cell lines. Their studies indicated that activation ofthe NFκB complex by overexpression of the p65 protein played a criticalrole in the process of thyroid cell transformation (Visconti et al.,Oncogene, 15, 16, 1987-1994 (1997)). Mukhopadhyay et al. investigatedthe expression of the p50 subunit of NFκB transcription factor complexin non-small cell lung carcinoma tissues demonstrating that 81% of freshnon-small cell lung cancer tissues express from two to twenty-foldhigher levels of the p50 subunit than normal lung tissue (Mukhopadhyayet al., Oncogene, 11, 5, 999-1003 (1995)). Khaled et al. targeted p50gene expression with specific antisense 3′ phosphorothioate modifiedantisense oligodeoxynucleotides and were able to reduce NFκB expression.Their results demonstrated p50 antisense molecules could reduce NFκBexpression and could down-regulate the immune response providingpossible treatment for autoimmune disorders (Khaled et al., Clin.Immunol. Immunopathol., 83, 3, 254-263 (1997)).

SUMMARY OF THE INVENTION

[0004] In accordance with the present invention, there are providednucleic acid sequences encoding nuclear factor κB inducing factor (NFIF)14b and 7a. Included also are cDNAs encoding NFIF-14b and NFIF-7a andisolated and purified NFIF-14b and NFIF-7a polypeptides which induceNFκB. The present invention includes also methods of inducing NFκBcomprising introducing into the body of a patient a composition thatactivates NFκB induction, including for example, expression vectors thatexpress NFIF-14b and NFIF-7a polypeptides. Examples of preferredexpression vectors are retroviral vectors, adenoviral vectors,adeno-associated viral vectors, herpes, herpesviral vectors and nakedDNA vectors. The present invention also provides methods wherein thecomposition of the present invention comprise an NFIF-14b or NFIF-7apolypeptide and a pharmaceutically acceptable carrier.

[0005] Another aspect of the present invention are compositions forlowering the expression of the NFIF gene comprising an antisense nucleicacid. Still another aspect of the present invention is a composition forlowering the activity of an NFIF polypeptide comprising a neutralizingantibody that binds to an NFIF polypeptide and lowers its activity.

[0006] Yet another embodiment of the present invention is a compositionfor lowering the expression of NFIF in a patient comprising a ribozymethat cuts RNA encoding an NFIF polypeptide.

[0007] The present invention provides methods for evaluating whether atest compound is effective in inhibiting the activity of NFIF-14bpolypeptides comprising (A) comparing the level of NFκB-regulated geneexpression in a first sample comprising: (1) NFIF-14b; (2) theNFκB-regulated reporter gene; and (3) the test compound with the levelof gene expression in a second sample comprising (4) NFIF-14b; and (5)the NFκB-regulated reporter gene; and (B) determining whether theexpression of the reporter gene is lower in the first sample relative tothe second sample.

[0008] Still another aspect of the present invention provides methodsfor evaluating whether a test compound is effective in inhibiting theactivity of NFIF-7a polypeptides comprising (A) comparing the level ofNFκB-regulated gene expression in a first sample comprising: (1)NFIF-7a; (2) the NFκB-regulated reporter gene; and (3) the test compoundwith the level of gene expression in a second sample comprising (4)NFIF-7a; and (5) the NFκB-regulated reporter gene; and (B) determiningwhether the expression of the reporter gene is lower in the first samplerelative to the second sample.

[0009] Yet another aspect of the present invention provides a method foridentifying whether a test compound can enhance the activity of NFIF-14bbased on the expression of an NFκB regulator reporter gene comprising(A) comparing the level of NFκB-regulated gene expression in a firstsample comprising (1) NFIF-14b; (2) the NFκB-regulated reporter gene;and (3) the test compound with the level of gene expression in a secondsample comprising: (4) NFIF-14b; and (5) the NFκB-regulated reportergene; and (B) determining whether the expression of the reporter gene ishigher in the first sample relative to the second sample.

[0010] Another aspect of the present invention provides a method foridentifying whether a test compound can enhance the activity of NFIF-7abased on the expression of an NFκB regulator reporter gene comprising(A) comparing the level of NFκB-regulated gene expression in a firstsample comprising (1) NFIF-7a; (2) the NFκB-regulated reporter gene; and(3) the test compound with the level of gene expression in a secondsample comprising: (4) NFIF-7a; and (5) the NFκB-regulated reportergene; and (B) determining whether the expression of the reporter gene ishigher in said first sample relative to the second sample.

[0011] In yet another aspect of the present invention, methods areprovided for inhibiting expression of NFκB-dependent genes comprisingadministering to a patient a composition that inhibits the activity ofNFIF-14b or NFIF-7a.

[0012] Still yet another aspect of the present invention is a method ofinhibiting inflammation comprising administration of a composition thatinhibits the activity of NFIF-14b or NFIF-7a.

[0013] Another aspect of the present invention relates to the use of anNFIF polypeptide for the manufacture of a medicament intended for thetreatment and/or prevention of an NFκB-regulated inflammatory response.Still yet another aspect of the present invention relates to the use ofa nucleic acid encoding an NFIF polypeptide, for the manufacture of amedicament intended for the treatment and/or prevention of anNFκB-regulated inflammatory response.

[0014] Another aspect of the present invention relates to the use of arecombinant vector comprising a nucleic acid encoding an NFIFpolypeptide, for the manufacture of a medicament intended for thetreatment and/or prevention of an NFκB-regulated inflammatory response.Yet another aspect of the present invention relates to the use of adefective recombinant viral vector for the manufacture of a medicamentintended for the treatment and/or prevention of an NFκB-regulatedresponse.

[0015] Another aspect of the present invention relates to the use ofcells genetically modified ex vivo with a recombinant virus orproduction of cells containing such recombinant viruses which areimplanted in the body, facilitating prolonged and effective expressionin vivo of an NFIF polypeptide according to the invention.

[0016] Two sequences demonstrating similarities to the polypeptides ofthe present invention are available in the GenBank nucleic acid sequencelibrary. The first sequence, having Accession Number Y08135 encodes apeptide identified as full-length mouse acid sphingomyelinase-likephosphodiesterase 3a. The second sequence, having Accession NumberY08136 encoded a partial human clone (863 bp of which 536 were coding)for a protein sequence identified as human acid sphingomyelinase-likephosphodiesterase 3a. Both sequences were submitted on Sep. 17, 1996 byK. Hofmann. Although both of these sequences were identified as encodingacid sphingomyelinase-like phosphodiesterases, there has not beenconfirmation of this type of enzymatic activity. These sequences mayhave been identified as acid sphingomyelinase-like phosphodiesterasesdue to similarities between the 3′ sequences of these proteins and the3′ sequences of members of the sphingomyelinase family.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is the amino acid sequence for NFIF-14b.

[0018]FIG. 2 is the amino acid sequence for NFIF-7a.

[0019]FIG. 3 is the cDNA sequence for NFIF-14b and NFIF-7a aligned forcomparison.

[0020]FIG. 4 is a diagrammatic representation of NFIF-14b and NFIF-7a.

[0021]FIG. 5 is a bar graph representing luminometer counts per secondobserved in HeK 293 cells at 48 hours.

[0022]FIG. 6 is a bar graph representing luminometer counts per secondobserved in Cos-7 cells at 48 hours.

[0023]FIG. 7 is the amino acid sequence of a 15-residue sequence used toprepare antibodies directed against NFIF.

[0024]FIG. 8 is a Northern Blot using an NFIF probe.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides polypeptides which are capable ofinducing NFκB as well as compounds and compositions that are capable ofinhibiting induction of NFκB.

[0026] The present invention is based in part on the discovery ofNFκB-inducing factor (NFIF) proteins. Two functional variants of theNFIF protein have been identified. One variant, NFIF-14b, comprisesfull-length NFIF. The second variant, NFIF-7a, is believed to be asplice variant of NFIF-14b. Both NFIF-14b and NFIF-7a have the abilityto induce NFκB.

[0027] NFκB comprises a family of eukaryotic transcription factors whichregulate genes involved in immune responses and other cellularactivities. In certain situations, it may be desirable to induce NFκB inorder to initiate or increase the extent of an immune response. In othersituations, it may be desirable to reduce or prevent NFκB induction inorder to reduce or prevent an NFκB-regulated immune response. Forexample, it has been discovered that NFIF is associated with a varietyof pathologies associated with NFκB-regulated immune responses,including atherosclerosis. By inhibiting the expression of the NFIFgene, or by otherwise interfering with the activity of the NFIF protein,it is possible to inhibit NFκB induction, thereby inhibiting orpreventing NFκB-regulated inflammatory responses that result inatherosclerosis and other diseases.

[0028] Discovery of the gene encoding NFIF and the ability to prepareNFIF proteins facilitates identification and preparation of compoundsand compositions which are capable of inhibiting the activity of NFIFproteins and consequently inhibiting NFκB induction.

[0029] The description which follows begins with a Definitions section,followed by background information relating to NFκB. This is followed bya discussion of the NFIF polypeptides of the present invention, as wellas information on isolation of DNA encoding such polypeptides andmethods for preparing variants of the polypeptides. Expression vectorscapable of expressing the encoding NFIF polypeptide DNAs are thendiscussed, including information on appropriate promoters for theexpression systems, methods for introducing the expression vectors intoappropriate host cells and viral vector systems. Following thisdiscussion of NFIF polypeptides and their variants, there is adiscussion of therapeutic uses for NFIF polypeptides. This discussion isfollowed by a description of compositions useful in the practice of thepresent invention. This is followed by a discussion which outlines thevarious therapeutic compositions and compounds, including polypeptidesbased on NFIF, antisense nucleic acids, ribozymes and antibodies.Finally, the methods of the present invention which use the describedcompounds and compositions are described.

[0030] Definitions

[0031] The term “NFIF” is used to designate “nuclear factor κB-inducingfactor”.

[0032] The terms “inducing” or “induction” when used in describing theactivity of NFIF include within their scope the ability to causeexpression of the subunit proteins which comprise NFκB either directlyor indirectly as well as the ability to activate NFκB that is alreadypresent in a cell. This activation may be direct or indirect and resultsin NFκB functioning as a transcription factor.

[0033] For the purposes of the present description, the expression“nucleotide sequence” may be used to designate either a polynucleotideor a nucleic acid. The expression “nucleotide sequence” covers thegenetic material itself and is therefore not restricted to theinformation relating to its sequence.

[0034] The terms “nucleic acid”, “polynucleotide”, “oligonucleotide” or“nucleotide sequence” cover RNA, DNA, gDNA or cDNA sequences oralternatively RNA/DNA hybrid sequences of more than one nucleotide,either in the single-stranded form or in the duplex, double-strandedform.

[0035] A “nucleic acid” is a polymeric compound comprised of covalentlylinked subunits called nucleotides. Nucleic acid includespolyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both ofwhich may be single-stranded or double-stranded. DNA includes cDNA,genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence ofnucleotides that encodes a protein is called the sense sequence orcoding sequence.

[0036] The term “nucleotide” designates both the natural nucleotides (A,T, G, C) as well as the modified nucleotides that comprise at least onemodification such as (1) an analog of a purine, (2) an analog of apyrimidine, or (3) an analogous sugar, examples of such modifiednucleotides being described, for example, in the PCT application No.WO95/04064.

[0037] For the purposes of the present invention, a first polynucleotideis considered as being “complementary” to a second polynucleotide wheneach base of the first nucleotide is paired with the complementary baseof the second polynucleotide whose orientation is reversed. Thecomplementary bases are A and T (or A and U), or C and G. “Heterologous”DNA refers to DNA not naturally located in the cell, or in a chromosomalsite of the cell. Preferably, the heterologous DNA includes a geneforeign to the cell.

[0038] As used herein, the term “homologous” in all its grammaticalforms and spelling variations refers to the relationship betweenproteins that possess a “common evolutionary origin,” including proteinsfrom superfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell, 50:667 (1987)). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity.

[0039] Accordingly, the term “sequence similarity” in all itsgrammatical forms refers to the degree of identity or correspondencebetween nucleic acid or amino acid sequences of proteins that may or maynot share a common evolutionary origin (see Reeck et al., supra).However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and not a common evolutionary origin.

[0040] In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and more preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., Molecular cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring, N.Y. (1982); Glover et al., DNACloning: A Practical Approach, Volumes I and II OligonucleotideSynthesis, MRL Press, Ltd., Oxford, U.K. (1985); Hames and Higgins,Hames B D and Higgins S J, 1985. Nucleic acid hybridization: a practicalapproach, Hames and Higgins Ed., IRL Press, Oxford (1985)).

[0041] Similarly, in a particular embodiment, two amino acid sequencesare “substantially homologous” or “substantially similar” when greaterthan 30% of the amino acids are identical, or greater than about 60% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program.

[0042] The “percentage identity” between two nucleotide or amino acidsequences, for the purposes of the present invention, may be determinedby comparing two sequences aligned optimally, through a window forcomparison.

[0043] The portion of the nucleotide or polypeptide sequence in thewindow for comparison may thus comprise additions or deletions (forexample “gaps”) relative to the reference sequence (which does notcomprise these additions or these deletions) so as to obtain an optimumalignment of the two sequences.

[0044] The percentage is calculated by determining the number ofpositions at which an identical nucleic base or an identical amino acidresidue is observed for the two sequences (nucleic or peptide) compared,and then by dividing the number of positions at which there is identitybetween the two bases or amino acid residues by the total number ofpositions in the window for comparison, and then multiplying the resultby 100 in order to obtain the percentage sequence identity.

[0045] The optimum sequence alignment for the comparison may be achievedusing a computer with the aid of known algorithms contained in thepackage from the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICSCOMPUTER GROUP (GCG), 575 Science Doctor, Madison, Wis.

[0046] By way of illustration, it will be possible to produce thepercentage sequence identity with the aid of the BLAST software(versions BLAST 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 andBLAST 2.0.6 of September 1998), using exclusively the default parameters(Altschul et al, J Mol. Biol., 215:403-410 (1990); Altschul et al,Nucleic Acids Res., 25:3389-3402 (1997)). Blast searches for sequencessimilar/homologous to a reference “request” sequence, with the aid ofthe Altschul et al. algorithm. The request sequence and the databasesused may be of the peptide or nucleic types, any combination beingpossible.

[0047] The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

[0048] “Variant” of a nucleic acid according to the invention will beunderstood to mean a nucleic acid that differs by one or more basesrelative to the reference polynucleotide. A variant nucleic acid may beof natural origin, such as an allelic variant that exists naturally, orit may also be a non-natural variant obtained, for example, by mutagenictechniques.

[0049] In general, the differences between the reference (generally,wild-type) nucleic acid and the variant nucleic acid are small such thatthe nucleotide sequences of the reference nucleic acid and of thevariant nucleic acid are very similar and, in many regions, identical.The nucleotide modifications present in a variant nucleic acid may besilent, which means that they do not alter the amino acid sequencesencoded by said variant nucleic acid.

[0050] However, the changes in nucleotides in a variant nucleic acid mayalso result in substitutions, additions or deletions in the polypeptideencoded by the variant nucleic acid in relation to the polypeptidesencoded by the reference nucleic acid. In addition, nucleotidemodifications in the coding regions may produce conservative ornon-conservative substitutions in the amino acid sequence of thepolypeptide.

[0051] Preferably, the variant nucleic acids according to the inventionencode polypeptides that substantially conserve the same function orbiological activity as the polypeptide of the reference nucleic acid oralternatively the capacity to be recognized by antibodies directedagainst the polypeptides encoded by the initial reference nucleic acid.

[0052] Some variant nucleic acids will thus encode mutated forms of thepolypeptides whose systematic study will make it possible to deducestructure-activity relationships of the proteins in question. Knowledgeof these variants in relation to the disease studied is essential sinceit makes it possible to understand the molecular cause of the pathology.

[0053] “Fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid “fragment” according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from 8, 10, 12, 15, 18,20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 consecutivenucleotides of a nucleic acid according to the invention.

[0054] A “nucleic acid molecule” refers to the phosphate ester polymericform of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

[0055] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., Molecular cloning: a laboratorymanual. 2ed. Cold Spring Harbor Laboratory, Cold spring Harbor, N.Y.(1989)). The conditions of temperature and ionic strength determine the“stringency” of the hybridization. For preliminary screening forhomologous nucleic acids, low stringency hybridization conditions,corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC. Hybridization requires that the two nucleicacids contain complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8). Preferably a minimum length for a hybridizable nucleic acidis at least about 10 nucleotides; preferably at least about 15nucleotides; and more preferably the length is at least about 20nucleotides.

[0056] In a specific embodiment, the term “standard hybridizationconditions” refers to a T_(m) of 55° C., and utilizes conditions as setforth above. In a preferred embodiment, the T_(m) is 60° C.; in a morepreferred embodiment, the T_(m) is 65° C.

[0057] “High stringency hybridization conditions” for the purposes ofthe present invention will be understood to mean the followingconditions:

[0058] 1-Membrane competition and PREHYBRIDIZATION:

[0059] Mix: 40 μl salmon sperm DNA (10 mg/ml)+40 μl human placental DNA(10 mg/ml)

[0060] Denature for 5 minutes at 96° C., then immerse the mixture inice.

[0061] Remove the 2×SSC and pour 4 ml of formamide mix in thehybridization tube containing the membranes.

[0062] Add the mixture of the two denatured DNAs.

[0063] Incubation at 42° C. for 5 to 6 hours, with rotation.

[0064] 2—Labeled probe competition:

[0065] Add to the labeled and purified probe 10 to 50 μl Cot I DNA,depending on the quantity of repeats.

[0066] Denature for 7 to 10 minutes at 95° C.

[0067] Incubate at 65° C. for 2 to 5 hours.

[0068] 3—HYBRIDIZATION:

[0069] Remove the prehybridization mix.

[0070] Mix 40 μl salmon sperm DNA+40 μl human placental DNA; denaturefor 5 min at 96° C., then immerse in ice.

[0071] Add to the hybridization tube 4 ml of formamide mix, the mixtureof the two DNAs and the denatured labeled probe/Cot I DNA.

[0072] Incubate 15 to 20 hours at 42° C., with rotation.

[0073] 4—Washes and Exposure:

[0074] One wash at room temperature in 2×SSC, to rinse.

[0075] Twice 5 minutes at room temperature 2×SSC and 0.1% SDS at 65° C.

[0076] Twice 15 minutes at 65° C. 1×SSC and 0.1% SDS at 65° C.

[0077] Envelope the membranes in clear plastic wrap and expose.

[0078] The hybridization conditions described above are adapted tohybridization, under high stringency conditions, of a molecule ofnucleic acid of varying length from 20 nucleotides to several hundredsof nucleotides. It goes without saying that the hybridization conditionsdescribed above may be adjusted as a function of the length of thenucleic acid whose hybridization is sought or of the type of labelingchosen, according to techniques known to one skilled in the art.Suitable hybridization conditions may, for example, be adjustedaccording to the teaching contained in the manual by Hames and Higgins(1985), supra or in the manual by F. Ausubel et al., Current Protocolsin Molecular Biology, Green Publishing Associates and Wileylnterscience, N.Y. (1989).

[0079] Nuclear Factor κB

[0080] Nuclear factor κB (NFκB) comprises a family of eukaryotictranscription factors. The NFκB transcription factors were firstidentified as factors which bound the enhancer elements in the κ lightchain gene in murine B lymphocytes. Subsequent studies demonstrated NFκBis found in almost all cells and regulates genes involved in tissueinflammation, cellular proliferation and cellular differentiation.

[0081] NFκB comprises at least five subunits: p50, p52, p65 (Re1A),c-Re1, and Re1B which can form homo- and heterodimers in variouscombinations. Active forms of NFκB are usually heterodimers comprised ofp65 (Re1A) and p50.

[0082] In most cells, NFκB exists as an inactive heterodimer which issequestered in the cytosol due to association with an inhibitory proteinI-κB. In response to stimuli such as inflammatory stimuli, the I-κBprotein is phosphorylated and degraded, resulting in disassociation ofthe I-κB-NFκB complex and translocation of NFκB into the nucleus. Oncein the nucleus, NFκB recognizes specific enhancer sites containing theNFκB DNA binding motif and interacts with basal transcription factors toinitiate RNA polymerase II mediated transcription in conjunction withthe TATA box binding protein. As mentioned above, this results in thetranscription of a wide variety of genes, particularly genes involved inimmune and inflammatory responses.

[0083] As mentioned above, the present invention is based in part on thediscovery of nuclear factor κB-inducing factor (NFIF) proteins. Theseproteins are involved in induction of NFκB. Discovery of these NFIFproteins provides different approaches to treating conditions thatinvolve NFκB-regulated inflammatory responses. NFκB-regulatedinflammatory responses are associated with a variety of disease stateswhich include, but are not limited to, rheumatoid arthritis,atherosclerosis, autoimmune diseases, viral diseases, NSAID-inducedgastropathy, neurodegenerative diseases, scrapie, sepsis, apoptosis,Crohn's disease, renal disease, restenosis, brain injury/inflammation,Alzheimer's disease, asthma, and improperly regulated expression ofpleiotropic cytokines. In situations where it is desirable to increaseinduction of NFκB for the purpose of resulting in an increased immuneresponse, the NFIF proteins of the present invention can be introducedto or expressed in a patient to induce NFκB. If, on the other hand, itis desirable to inhibit induction of NFκB in order to inhibit or preventan immune response, the provision according to the present invention ofthe genetic sequences encoding the NFIF proteins and of how to preparethese proteins can be used to identify compounds and compositions whichinhibit or prevent expression of the NFIF proteins or which interactwith NFIF proteins to inhibit or prevent their activity.

[0084] Nuclear Factor κB Inducing Factor (NFIF) Proteins

[0085] The polypeptides and proteins of the present invention includerecombinant polypeptides, natural polypeptides, or syntheticpolypeptides, and can be of human, rabbit, or other animal origin.

[0086] The polypeptides can be isolated from natural sources, such asplacental extracts, human plasma, or conditioned media from culturedcells using purification procedures known to one of skill in the art.

[0087] Alternatively, the polypeptides of the present invention can beprepared utilizing recombinant DNA technology which comprises combininga nucleic acid encoding the polypeptide thereof in a suitable vector,inserting the resulting vector into a suitable host cell, recovering thepolypeptide produced by the resulting host cell, and purifying thepolypeptide recovered.

[0088] The polypeptides are characterized by a reproducible singlemolecular weight and/or multiple set of molecular weights,chromatographic properties and elution profiles, amino acid compositionand sequence, and biological activity.

[0089] Isolation of Nucleotide Sequences Encoding NFIF Polypeptides

[0090] The NFIF-14b and NFIF-7a embodiments of the present invention canbe prepared by a variety of suitable methods known to those of skill inthe art. Teachings on general molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); F. M. Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0091] The cDNA sequence for NFIF-14b and NFIF-7a are presented in FIG.3. NFIF-7a is a splice variant of NFIF-14b. The term “splice variant”refers to a polypeptide encoded by an mRNA produced by alternativeprocessing of the full length mRNA encoded by a gene or genes resultingin an mRNA that contains one or more deletions relative to the fulllength mRNA for the genes. As shown in FIG. 4, relative to NIFI-14b,NFIF-7a has an internal deletion of base pairs 473 to 739.Given theinformation in the description herein on the DNA sequence of NFIF-14band NFIF-7a and the known methods in the art for obtaining cDNA,nucleotide sequences encoding NFIF-14b and NFIF-7a can be cloned readilyand inserted into an appropriate vector for expression of these proteinsin vitro or in vivo. For a description of methods relating to cloningcDNA and expression vectors, see Sambrook et al., 1989, supra. A“cloning vector” is a replicon, for example, a plasmid, phage or cosmid,to which another DNA segment may be attached so as to bring about thereplication of the attached segment. A “replicon” is any genetic element(e.g., plasmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. A cloning vector may be capable of replication in one cell typeand expression in another (“shuttle vector”). In preferred embodimentsof the present invention, the cloning vector is capable of expression ina host cell and the “expression vector” is able to express NFIF atsufficient levels to effect an NFκB regulated pathway in the cell.

[0092] A gene encoding NFIF-14b and NFIF-7a, whether genomic DNA orcDNA, can be isolated from a human genomic library or cDNA library. ADNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The DNA coding sequences and the appropriate regulatory sequences arepreferably provided in an expression vector. The boundaries of thecoding sequence are determined by a start codon at the 5' (amino)terminus and a translation stop codon at the 3' (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Ifthe coding sequence is intended for expression in a eukaryotic cell, apolyadenylation signal and transcription termination sequence willusually be located 3' to the coding sequence. Methods for obtaining agene given the DNA sequence information presented herein are well knownin the art. The DNA may be obtained by standard procedures known in theart from cloned DNA (e.g., a DNA “library”). It is obtained preferablyfrom a cDNA library prepared from tissues with high level expression ofthe protein. The DNA may also be obtained by the cloning of genomic DNA,or fragments thereof, purified from the desired cell (See, for example,Sambrook et al., 1989, supra; Glover, D. M. (ed.), 1985, DNA Cloning: APractical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II) or bychemical synthesis. Clones derived from genomic DNA may containregulatory and intron DNA regions in addition to coding regions.

[0093] Methods for obtaining cDNA are well known in the art. Briefly,these methods include isolating a mixture of messenger RNA (mRNAs) fromeukaryotic cells and employing a series of enzymatic reactions tosynthesize double-stranded DNA copies (cDNAs) complementary to theisolated mRNAs. “Polymerase chain reaction” (PCR) refers to in vitromethods for amplifying specific DNA sequences using techniques wellknown in the art.

[0094] Regardless of the method used to obtain the desired cDNA, thedouble-stranded cDNA mixture is inserted into cloning vehicles by anyone of many known techniques, depending at least in part on theparticular vehicle used. Various insertion methods are discussed inSambrook et al., 1989, supra and are well known in the art. A “cassette”refers to a segment of DNA that can be inserted into a vector at one ormore specific restriction sites. The segment of DNA encodes apolypeptide of interest and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

[0095] Once the DNA segments are inserted into a cloning vehicle, thecloning vehicle is used to transform a suitable host. A cell has been“transfected” by exogenous or heterologous DNA when such DNA has beenintroduced inside the cell. A cell has been “transformed” by exogenousor heterologous DNA when the transfected DNA effects a phenotypicchange. The transforming DNA can be integrated (covalently linked) intochromosomal DNA making up the genome of the cell. These cloning vehiclesusually impart an antibiotic resistance trait on the host. Such hostsare generally prokaryotic cells and only a few of the host cells containthe desired cDNA. The transfected host cells constitute a gene“library”, providing a representative sample of the mRNAs present in thecell from which the mRNAs were isolated.

[0096] Given the sequence information on NFIF-14b and NFIF-7a providedherein, an appropriate oligonucleotide may be prepared, preferablysynthesized as discussed above, and used to identify clones containingNFIF sequences. The oligonucleotide preferably includes at least about18 nucleotides and is hybridizable to a genomic DNA molecule, a cDNAmolecule, or an mRNA molecule encoding NFIF. Oligonucleotides can belabeled, e.g., with ³²P-nucleotides or nucleotides to which a label,such as biotin, has been covalently conjugated.

[0097] In one embodiment, a labeled oligonucleotide can be used as aprobe to detect the presence of a nucleic acid encoding NFIF. In anotherembodiment, oligonucleotides (one or both of which may be labeled) canbe used as PCR primers, either for cloning full length or a fragment ofNFIF, or to detect the presence of nucleic acids encoding NFIF. In afurther embodiment, an oligonucleotide can form a triple helix with anNFIF DNA molecule.

[0098] Generally, oligonucleotides are prepared synthetically,preferably on a nucleic acid synthesizer. Accordingly, oligonucleotidescan be prepared with non-naturally occurring phosphoester analog bonds,such as thioester bonds, etc. To identify clones containing the NFIFsequences, individual transformed or transfected cells are grown ascolonies on a nitrocellulose filter paper. The colonies are lysed andthe DNA is bound tightly to the filter paper by heating. The filterpaper is then incubated with a labeled oligonucleotide probe which iscomplementary to NFIF. DNA fragments with substantial homology to NFIFwill hybridize to the probe.

[0099] As discussed above, the conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Hybridizationrequires that the two nucleic acids contain complementary sequences,although depending on the stringency of the hybridization, mismatchesbetween bases are possible. The appropriate stringency for hybridizingnucleic acids depends on the length of the nucleic acids and the degreeof complementation, variables well known in the art.

[0100] The probe hybridizes with the cDNA for which it is complementary.It can be identified by autoradiography or by chemical reactions thatidentify the presence of the probe. The corresponding clones arecharacterized in order to identify one or a combination of clones whichcontain all of the structural information for the desired protein. Thenucleic acid sequence coding for the protein of interest is isolated andreinserted into an expression vector. The expression vector brings thecloned gene under the regulatory control of specific prokaryotic oreukaryotic control elements which allow the efficient expression(transcription and translation) of the ds-cDNA. Transcriptional andtranslational control sequences are DNA regulatory sequences, such as,for example, promoters, enhancers, and terminators that provide for theexpression of a coding sequence in a host cell. In eukaryotic cells,polyadenylation signals are control sequences. A coding sequence is“under the control” of transcriptional and translational controlsequences in a cell when RNA polymerase transcribes the coding sequenceinto mRNA, which is then spliced (if the coding sequence containsintrons) and translated into the protein encoded by the coding sequence.

[0101] Further selection can be carried out on the basis of theproperties of the gene. For example, the presence of the desired gene ina clone may be detected by assays based on the physical, chemical, orimmunological properties of its expressed protein product. For example,cDNA clones, or DNA clones can be selected which produce a protein thathas similar or identical properties to those of NFIF with regard toelectrophoretic migration, isoelectric focusing, non-equilibrium pH gelelectrophoresis, proteolytic digestion, or antigenicity.

[0102] Preparation of Variants of NFIF Polypeptides

[0103] The present invention includes within its scope allelic variants,substitution, addition and deletion mutant variants, analogs, andderivatives of NFIF and homologs from other species that have the sameor homologous functional activity as NFIF. In preferred embodiments,genes having deletions or substitutions that increase the ability toinduce NFκB are utilized in the practice of the invention. Preparationor isolation of NFIF variants are within the scope of the presentinvention. Accordingly, the scope of the present invention includes NFIFvariants which are functionally active, i.e., capable of exhibiting oneor more functional activities associated with NFIF.

[0104] NFIF variants can be made by altering encoding nucleic acidsequences by substitutions, additions or deletions that provide forfunctionally equivalent molecules. Preferably, NFIF embodiments are madethat have enhanced or increased functional activity relative to NFIF-14bor NFIF-7a.

[0105] Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence asNFIF, including an amino acid sequence that contains a single amino acidvariant, can be used in the practice of the present invention. Theseinclude, but are not limited to, allelic genes, homologous genes fromother species, and nucleotide sequences comprising all or portions ofNFIF which are altered by the substitution of different codons thatencode the same amino acid residue within the sequence, thus producing asilent change. Likewise, the NFIF variants of the invention include, butare not limited to, those containing, as a primary amino acid sequence,all or part of the amino acid sequence of a NFIF protein includingaltered sequences in which functionally equivalent amino acid residuesare substituted for residues within the sequence resulting in aconservative amino acid substitution. For example, one or more aminoacid residues within the sequence can be substituted by another aminoacid of a similar polarity, which acts as a functional equivalent,resulting in a silent alteration. Substitutes for an amino acid withinthe sequence can be selected from other members of the class to whichthe amino acid belongs. For example, the nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. Amino acids containingaromatic ring structures are phenylalanine, tryptophan, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point.

[0106] Particularly preferred substitutions are:

[0107] Lys for Arg and vice versa such that a positive charge may bemaintained;

[0108] Glu for Asp and vice versa such that a negative charge may bemaintained;

[0109] Ser for Thr such that a free —OH can be maintained; and

[0110] Gln for Asn such that a free CONH₂ can be maintained.

[0111] Amino acid substitutions may also be introduced to substitute anamino acid with a particularly preferable property. For example, a Cysmay be introduced as a potential site for disulfide bridges with anotherCys. A His may be introduced as a particularly “catalytic” site (i.e.,His can act as an acid or base and is the most common amino acid inbiochemical catalysis). Pro may be introduced because of itsparticularly planar structure, which induces β-turns in the protein'sstructure.

[0112] The genes encoding NFIF variants of the invention can be producedby various methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example,the cloned NFIF gene sequence can be modified by any of numerousstrategies known in the art (Sambrook et al., 1989, supra). The sequencecan be cleaved at appropriate sites with restriction endonuclease(s),followed by further enzymatic modification if desired, isolated, andligated in vitro. In the production of the gene encoding a NFIFembodiment, care should be taken to ensure that the modified generemains within the same translational reading frame as the NFIF gene,uninterrupted by translational stop signals, in the gene region wherethe desired activity is encoded.

[0113] Additionally, the NFIF-encoding nucleic acid sequence can bemutated in vitro or in vivo to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification.Preferably, such mutations enhance the functional activity of themutated NFIF gene product. Any technique for mutagenesis known in theart can be used, for example, in vitro site-directed mutagenesis(Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551; Zoller andSmith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177;Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), and useof “TAB” linkers (Pharmacia), etc. PCR techniques are preferred for sitedirected mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA” inPCR Technology: Principles and Applications for DNA Amplification, H.Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

[0114] Polypeptides Based on NFIF

[0115] A therapeutically useful variant of NFIF may be identified by avariety of methods, including in vitro assays that can be used toidentify the level of expression of an NFκB regulated gene or a genewhose expression is regulated by NFκB regulatory elements in thepresence of the NFIF variant. One method for identifying polypeptideswhich are able to enhance (or inhibit) NFκB induction is to use areporter gene system. These systems utilize reporter gene expressionvectors which include a cloning site into which a given promoter may becloned upstream of a “reporter gene” which can be easily detected andquantified. One of skill in the art could readily identify and subclonethe promoter for NFκB as well as other control sequences into acommercially available reporter gene expression vector. The expressionvector is transferred into host cells and the cells are exposed to anNFIF variant (or a putative inhibitor or enhancer molecule) to determinethe effect on expression of the reporter gene product. In particular,the cells are assayed for the presence of the reporter gene productdirectly by measuring the amount of reporter mRNA, the reporter proteinitself or the enzymatic activity of the reporter protein. Ideally, thereporter gene is not endogenously expressed in the cell type of interestand lends itself to sensitive, quantitative and rapid assays. A varietyof reporter assay constructs are commercially available and severalreporter genes and assays have been developed and can be readilyprepared by those of skill in the art. The most popular systems formonitoring genetic activity in eukaryotic cells include chloramphenicolacetyltransferase (CAT), β-galactosidase, firefly luciferase, growthhormone (GH), β-glucurorudase (GUS), alkaline phosphatase (AP), greenfluorescent protein (GFP) and Renilla luciferase. Reporter assayconstructs can be purchased from a variety of sources including Promegaand Invitrogen.

[0116] As mentioned above, reporter gene activity can be detected byassaying for the reporter mRNA or the reporter protein. The reportermRNA can be detected by northern blot analysis, ribonuclease protectionassays or RT-PCR. While these assays are more direct than measuringprotein expression, many assays have been developed to measure thepresence of the reporter protein rather than the mRNA present in a cell.Reporter proteins can be assayed by spectrophotometry or by detectingenzymatic activity. Reporter protein levels may also be measured withantibody-based assays. In general, the enzymatic assays are verysensitive and are a preferred method of monitoring reporter geneexpression. A preferred commercially available NFκB reporter geneconstruct is the pNFκB-Luc (luciferase) reporter gene vector availablefrom Stratagene. An example of how to utilize this reporter system toquantify NFIF protein activity is provided in Example 4.

[0117] Experiments of the type discussed hereinabove can be utilized todetermine how well a given disease state can be treated using thecompositions of the present invention.

[0118] The discussion which follows relates to the manipulation andexpression of DNA encoding the polypeptides of the present invention.

[0119] Expression Vectors Encoding NFIF Polypeptides

[0120] The identified and isolated DNA sequence can be inserted into anappropriate cloning/expression vector (hereinafter “vector”) tofacilitate modifications to the sequence or expression of the protein.These vectors typically include multiple cloning sites, promoters,sequences which facilitate replication in a host cell and selectionmarkers.

[0121] Any suitable vector can be used. There are many known in the art.Examples of vectors that can be used include, for example, plasmids ormodified viruses. The vector is typically compatible with a given hostcell into which the vector is introduced to facilitate replication ofthe vector and expression of the encoded proteins. The insertion of aDNA sequence into a given vector can, for example, be accomplished byligating the DNA fragment into a cloning vector which has complementarycohesive termini. However, if the complementary restriction sites usedto cut the DNA are not present in the cloning vector, the ends of theDNA molecules may be enzymatically modified. Alternatively, any sitedesired may be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; the ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. Useful vectors may consist of segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Examples ofspecific vectors useful in the practice of the present invention are E.coli bacteriophages, for example, lambda derivatives, or plasmids, forexample, pBR322 derivatives or pUC plasmid derivatives, e.g., pmal-c,pFLAG, derivatives of SV40 and known bacterial plasmids, e.g., E. coliplasmids col E1, pCR1, pMal-C2, pET, pGEX (Smith et al., 1988, Gene67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAs,e.g., the numerous derivatives of phage 1, e.g., NM989, and other phageDNA, e.g., M13 and filamentous single stranded phage DNA; yeast vectorssuch as the 2 μm plasmid or derivatives thereof; vectors useful ineukaryotic cells, for example, vectors useful in insect cells, such asbaculovirus vectors, vectors useful in mammalian cells; vectors derivedfrom combinations of plasmids and phage DNAs, plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like.

[0122] Examples of yeast vectors that can be used according to theinvention are the non-fusion pYES2 vector (Invitrogen) or the fusionpYESHisA, B, C (Invitrogen).

[0123] Baculovirus vectors that can be used in the practice of theinvention include a variety of vectors, including both non-fusiontransfer vectors, for example, pVL941 (Summers), pVL1393 (Invitrogen),pVL1392 (Summers and Invitrogen), and pBlueBacIII (Invitrogen), andfusion transfer vectors, for example, pAc700 (Summers), pAc701 andpAc702 , pAc360 (Invitrogen), and pBlueBacHisA, B, C (Invitrogen) can beused.

[0124] Mammalian vectors contemplated for use in the invention include,for example, vectors with inducible promoters, for example, thedihydrofolate reductase (DHFR) promoter, e.g., any expression vectorwith a DHFR expression vector, or a DHFR/methotrexate co-amplificationvector, for example, pED (see Kaufman, Current Protocols in MolecularBiology, 16.12 (1991)). Alternatively, a glutamine synthetase/methioninesulfoximine co-amplification vector, for example, pEE14 (Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, for example, pREP4(Invitrogen), pCEP4 (Invitrogen), pMEP4 (Invitrogen), pREP8(Invitrogen), pREP9 (Invitrogen), and pEBVHis (Invitrogen). Selectablemammalian expression vectors for use in the invention include pRc/CMV(Invitrogen), pRc/RSV (Invitrogen), pcDNA3 (Invitrogen) and others.Vaccinia virus mammalian expression vectors (see, Kaufman, 1991, supra)for use according to the invention include but are not limited topSCl11, pMJ601, and pTKgptF1S.

[0125] A variety of methods may be used to confirm that the desired DNAsequence encoding NFIF-14b, NFIF-7a, or another NFIF variant, has beencloned into a vector. In general, one or more of the followingapproaches is used: (a) PCR amplification of the desired plasmid DNA orspecific mRNA, (b) nucleic acid hybridization, (c) presence or absenceof selection marker gene functions, (d) analyses with appropriaterestriction endonucleases, and (e) expression of inserted sequences. Inthe first approach, the nucleic acids can be amplified by PCR to providefor detection of the amplified product. In the second approach, thepresence of a foreign gene inserted in an expression vector can bedetected by nucleic acid hybridization using probes comprising sequencesthat are homologous to an inserted marker gene. In the third approach,the recombinant vector/host system can be identified and selected basedupon the presence or absence of certain “selection marker” genefunctions (e.g., β-galactosidase activity, thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of foreign genesin the vector. In another example, if the nucleic acid encoding NFIF isinserted within the “selection marker” gene sequence of the vector,recombinants containing the NFIF insert can be identified by the absenceof the selection marker gene function. In the fourth approach,recombinant expression vectors are identified by digestion withappropriate restriction enzymes. In the fifth approach, recombinantexpression vectors can be identified by assaying for the activity,biochemical, or immunological characteristics of the gene productexpressed by the recombinant, provided that the expressed proteinassumes a functionally active conformation.

[0126] Promoters

[0127] The nucleotide sequence coding for NFIF-14b or NFIF-7a or a NFIFvariant thereof can be inserted into an expression vector which containsthe necessary elements for the transcription and translation of theinserted protein-coding sequence. Such elements are termed herein a“promoter.”

[0128] A promoter is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

[0129] The nucleic acid encoding the polypeptides of the invention isoperationally associated with a promoter in an expression vector of theinvention. Both cDNA and genomic sequences can be cloned and expressedunder control of such regulatory sequences. An expression vector alsopreferably includes a replication origin. The necessary transcriptionaland translational signals can be provided on a recombinant expressionvector or they may be supplied by the native gene encoding NFIF and/orits flanking regions. Any of the methods previously described for theinsertion of DNA fragments into a cloning vector may be used toconstruct expression vectors containing a gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombination (genetic recombination).

[0130] Expression may be controlled by any promoter/enhancer elementknown in the art, but these regulatory elements must be functional inthe host selected for expression. Examples of promoters which may beused to control NFIF gene expression include the SV40 early promoterregion (Benoist and Chambon, 1981, Nature 290:304-310), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors for example, the β-lactamase promoter (Villa-Kamaroff, et al.,1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25);promoter elements from yeast or other fungi, for example, the Gal 4promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerolkinase) promoter, alkaline phosphatase promoter; and the animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.,50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grossched1 et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.,7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol., 5:1639-1648; Hammer et al., 1987, Science 235:53-58),alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel., 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

[0131] A preferred promoter used in the expression vector constructsdiscussed hereinbelow is the cytomegalovirus (CMV) promoter which iscapable of providing high level expression in a variety of mammaliancell lines.

[0132] Introduction of Vectors into Host Cells

[0133] Vectors can be introduced into host cells by any suitable method,including, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990) sothat many copies of the gene sequence are generated. In a preferredmethod, cells are transfected in vitro utilizing Lipfectamine® availablefrom Gibco-BRL. Preferably, the cloned gene is contained on a shuttlevector plasmid, which provides for expansion in a cloning cell, e.g., E.coli, and facilitates purification for subsequent insertion into anappropriate expression cell line. For example, a shuttle vector, whichis a vector that can replicate in more than one type of organism, can beprepared for replication in both E. coli and Saccharomyces cerevisiae bylinking sequences from an E. coli plasmid with sequences from the yeast2 μm plasmid.

[0134] Host Cell Systems

[0135] Host cell systems include mammalian host cell systems, insecthost cell systems and microorganisms such as yeast or bacteria.Depending on the host cell system utilized, any one of a number ofsuitable transcription and translation elements may be used.

[0136] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the desiredmodification and processing of the foreign protein expressed. Expressionin yeast can produce a biologically active product. Expression ineukaryotic cells can increase the likelihood of “native” folding.Moreover, expression in mammalian cells can provide a tool forreconstituting, or constituting, NFIF-inhibiting activity. Furthermore,different vector/host expression systems may affect processingreactions, such as proteolytic cleavages, to a different extent.Expression vectors of the invention can be used, as pointed out above,both to transfect cells for screening or biological testing ofmodulators of NFIF activity.

[0137] Examples of acceptable mammalian host cells are HEK 293 cells andCOS-7 cells.

[0138] A recombinant NFIF-14b, NFIF-7a or NFIF variant of the inventionmay be expressed chromosomally, after integration of the coding sequenceby recombination. In this regard, any of a number of amplificationsystems may be used to achieve high levels of stable gene expression(See Sambrook et al., 1989, supra).

[0139] The cell into which the recombinant vector comprising the nucleicacid encoding NFIF is introduced is cultured in an appropriate cellculture medium under conditions that provide for expression of an NFIFpolypeptide by the cell.

[0140] Once a suitable host system and growth conditions areestablished, recombinant expression vectors can be propagated andprepared in quantity. Soluble forms of the protein can be obtained bycollecting culture fluid, or solubilizing inclusion bodies, e.g., bytreatment with detergent, and if desired sonication or other mechanicalprocesses, as described above. The solubilized or soluble protein can beisolated using various techniques, including polyacrylamide gelelectrophoresis (PAGE), isoelectric focusing, 2-dimensional gelelectrophoresis, chromatography (e.g., ion exchange, affinity,immunoaffinity, and sizing column chromatography), centrifugation,differential solubility, immunoprecipitation, or by any other standardtechnique for the purification of proteins.

[0141] As discussed above, a “vectors” is any means for the transfer ofa nucleic acid according to the invention into a host cell. Preferredvectors are viral vectors, for example, retroviruses, herpes viruses,adenoviruses, and adeno-associated viruses. Thus, a gene encoding aprotein or polypeptide domain fragment of the present invention isintroduced in vivo, ex vivo, or in vitro using a viral vector or throughdirect introduction of DNA. Expression in targeted tissues can beeffected by targeting the transgenic vector to specific cells, such aswith a viral vector or a receptor ligand, or by using a tissue-specificpromoter, or both.

[0142] The discussion which follows outlines various viral and non-viralsystems that may be used to introduce DNA into a host cell in vivo or invitro.

[0143] Viral Vector Systems

[0144] The NFIF polypeptides, as well as the antisense nucleic acids,ribozymes and antibodies discussed hereinbelow may be prepared in vitroor ex vivo or may be designed to be expressed in vivo in a patient usingan appropriate expression system introduced via a viral vector system.

[0145] Viral vectors commonly used for in vivo or ex vivo targeting andtherapy procedures are DNA-based vectors and retroviral vectors. Methodsfor constructing and using viral vectors are known in the art [see,e.g., Miller and Rosman, BioTechniques 7:980-990 (1992)]. Preferably,the viral vectors are replication defective, that is, they are unable toreplicate autonomously in the target cell. In general, the genome of thereplication defective viral vectors which are used within the scope ofthe present invention lack at least one region which is necessary forthe replication of the virus in the infected cell. These regions caneither be eliminated (in whole or in part), or be renderednon-functional by any technique known to a person skilled in the art.These techniques include the total removal, substitution (by othersequences, in particular by the inserted nucleic acid), partial deletionor addition of one or more bases to an essential (for replication)region. Such techniques may be performed in vitro (on the isolated DNA)or in situ, using the techniques of genetic manipulation or by treatmentwith mutagenic agents. Preferably, the replication defective virusretains the sequences of its genome which are necessary forencapsulating the viral particles.

[0146] DNA viral vectors include an attenuated or defective DNA virus,such as but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV),vaccinia virus, and the like. Defective viruses, which entirely oralmost entirely lack viral genes, are preferred. Defective virus is notreplication competent after introduction into a cell, and thus does notlead to a productive viral infection. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Thus, a specifictissue can be specifically targeted. Examples of particular vectorsinclude, but are not limited to, a defective herpes virus 1 (HSV 1)vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330 (1991)),defective herpes virus vector lacking a glyco-protein L gene (PatentPublication RD 371005 A), or other defective herpes virus vectors(International Patent Publication No. WO94/21807, published Sep. 29,1994; International Patent Publication No. WO92/05263, published Apr. 2,1994); an attenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. (J. Clin. Invest. 90:626-630 (1992); seealso La Salle et al., Science 259:988-990 (1993)); and a defectiveadeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101(1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski etal., Mol. Cell. Biol. 8:3988-3996 (1988)]) Preferably, for in vivoadministration, an appropriate immunosuppressive treatment is employedin conjunction with the viral vector, e.g., adenovirus vector, to avoidimmuno-deactivation of the viral vector and transfected cells. Forexample, immunosuppressive cytokines, such as interleukin-12 (IL-12),interferon-γ (IFN-γ), or anti-CD4 antibody, can be administered to blockhumoral or cellular immune responses to the viral vectors. In addition,it is advantageous to employ a viral vector that is engineered toexpress a minimal number of antigens.

[0147] Naturally, the invention contemplates delivery of a vector thatwill express a therapeutically effective amount of NFIF for gene therapyapplications. The phrase “therapeutically effective amount” is usedherein to mean an amount sufficient to cause an improvement in aclinically significant condition in the host.

[0148] Certain viral vector systems are well developed in the art andare suited to the treatment methods of the present invention.

[0149] Adenovirus Vector Systems

[0150] In a preferred embodiment, the vector is an adenovirus vector.Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to usingtype 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses ofanimal origin (see WO94/26914). Those adenoviruses of animal originwhich can be used within the scope of the present invention includeadenoviruses of canine, bovine, murine (example: Mav1, Beard et al.,Virology 75 (1990) 81), bovine, porcine, avian, and simian (example:SAV) origin.

[0151] Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1region of theadenoviral vector is non-functional. The deletion in the E1 regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAd5 adenovirus (PvuII-Bg1II fragment) or 382 to 3446 (HinfII-Sau3Afragment). Other regions may also be modified, in particular the E3region (WO95/02697), the E2 region (WO94/28938), the E4 region(WO94/28152, WO94/12649 and WO95/02697), or in any of the late genesL1-L5.

[0152] In a preferred embodiment, the adenoviral vector has a deletionin the E1 region (Ad 1.0). Examples of E1-deleted adenoviruses aredisclosed in EP 185,573, the contents of which are incorporated hereinby reference. In another preferred embodiment, the adenoviral vector hasa deletion in the E1 and E4 regions (Ad 3.0). Examples of E1/E4-deletedadenoviruses are disclosed in WO95/02697 and WO96/22378, the contents ofwhich are incorporated herein by reference. In still another preferredembodiment, the adenoviral vector has a deletion in the E1 region intowhich the E4 region and the nucleic acid sequence are inserted (see FR9413355, the contents of which are incorporated herein by reference).

[0153] The replication defective recombinant adenoviruses according tothe invention can be prepared by any technique known to the personskilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573;Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared byhomologous recombination between an adenovirus and a plasmid whichcarries, inter alia, the DNA sequence of interest. The homologousrecombination is effected following cotransfection of the adenovirus andplasmid into an appropriate cell line. The cell line which is employedshould preferably (i) be transformable by the said elements, and (ii)contain the sequences which are able to complement the part of thegenome of the replication defective adenovirus, preferably in integratedform in order to avoid the risks of recombination. Examples of celllines which may be used are the human embryonic kidney cell line 293(Graham et al., J. Gen. Virol. 36 (1977) 59) which contains theleft-hand portion of the genome of an Ad5 adenovirus (12%) integratedinto its genome, and cell lines which are able to complement the E1 andE4 functions, as described in applications WO94/26914 andWO95/02697.Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques, which are well known to one ofordinary skill in the art.

[0154] Adeno-Associated Virus Vector Systems

[0155] The adeno-associated viruses (AAV) are DNA viruses of relativelysmall size which can integrate, in a stable and site-specific manner,into the genome of the cells which they infect. They are able to infecta wide spectrum of cells without inducing any effects on cellulargrowth, morphology or differentiation, and they do not appear to beinvolved in human pathologies. The AAV genome has been cloned, sequencedand characterised. It encompasses approximately 4700 bases and containsan inverted terminal repeat (ITR) region of approximately 145 bases ateach end, which serves as an origin of replication for the virus. Theremainder of the genome is divided into two essential regions whichcarry the encapsulation functions: the left-hand part of the genome,which contains the rep gene involved in viral replication and expressionof the viral genes; and the right-hand part of the genome, whichcontains the cap gene encoding the capsid proteins of the virus.

[0156] The use of vectors derived from the AAVs for transferring genesin vitro and in vivo has been described (see WO91/18088; WO93/09239;U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528). These publicationsdescribe various AAV-derived constructs in which the rep and/or capgenes are deleted and replaced by a gene of interest, and the use ofthese constructs for transferring the gene of interest in vitro (intocultured cells) or in vivo, (directly into an organism). The replicationdefective recombinant AAVs according to the invention can be prepared bycotransfecting a plasmid containing the nucleic acid sequence ofinterest flanked by two AAV inverted terminal repeat (ITR) regions, anda plasmid carrying the AAV encapsulation genes (rep and cap genes), intoa cell line which is infected with a human helper virus (for example anadenovirus). The AAV recombinants which are produced are then purifiedby standard techniques.

[0157] The invention also relates, therefore, to an AAV-derivedrecombinant virus whose genome encompasses a sequence encoding a nucleicacid encoding NFIF or its variants flanked by the AAV ITRs. Theinvention also relates to a plasmid encompassing a sequence encoding anucleic acid encoding NFIF or its variants flanked by two ITRs from anAAV. Such a plasmid can be used as it is for transferring the nucleicacid sequence, with the plasmid, where appropriate, being incorporatedinto a liposomal vector (pseudo-virus).

Retrovirus Vector Systems

[0158] In another embodiment the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol.62:1120; Temin et al., U.S. Pat. No. 5,124,263; EP 453242, EP178220;Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology3(1985) 689; International Patent Publication No. WO 95/07358, publishedMar. 16, 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845.The retroviruses are integrating viruses which infect dividing cells.The retrovirus genome includes two LTRs, an encapsulation sequence andthree coding regions (gag, pol and env). In recombinant retroviralvectors, the gag, pol and env genes are generally deleted, in whole orin part, and replaced with a heterologous nucleic acid sequence ofinterest. These vectors can be constructed from different types ofretrovirus, such as, HIV, MoMuLV (“murine Moloney leukaemia virus” MSV(“murine Moloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV(“spleen necrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus.Defective retroviral vectors are disclosed in WO95/02697.

[0159] In general, in order to construct recombinant retrovirusescontaining a nucleic acid sequence, a plasmid is constructed whichcontains the LTRs, the encapsulation sequence and the coding sequence.This construct is used to transfect a packaging cell line, which cellline is able to supply in trans the retroviral functions which aredeficient in the plasmid. In general, the packaging cell lines are thusable to express the gag, pol and env genes. Such packaging cell lineshave been described in the prior art, in particular the cell line PA317(U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO90/02806) and theGP+envAm-12 cell line (WO89/07150). In addition, the recombinantretroviral vectors can contain modifications within the LTRs forsuppressing transcriptional activity as well as extensive encapsulationsequences which may include a part of the gag gene (Bender et al., J.Virol. 61 (1987) 1639). Recombinant retroviral vectors are purified bystandard techniques known to those having ordinary skill in the art.

[0160] Retroviral vectors can be constructed to function as infectionsparticles or to undergo a single round of transfection. In the formercase, the virus is modified to retain all of its genes except for thoseresponsible for oncogenic transformation properties, and to express theheterologous gene. Non-infectious viral vectors are prepared to destroythe viral packaging signal, but retain the structural genes required topackage the co-introduced virus engineered to contain the heterologousgene and the packaging signals. Thus, the viral particles that areproduced are not capable of producing additional virus.

[0161] Non-Viral Systems

[0162] Certain non-viral systems have been used in the art and canfacilitate introduction of DNA encoding the NFIF polypeptides, antisensenucleic acids, ribozymes and antibodies.

Lipofection Delivery Systems

[0163] A vector can be introduced in vivo by lipofection. For the pastdecade, there has been increasing use of liposomes for encapsulation andtransfection of nucleic acids in vitro. Synthetic cationic lipidsdesigned to limit the difficulties and dangers encountered with liposomemediated transfection can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner, et. al., Proc. Natl.Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey, et al., Proc. Natl.Acad. Sci. U.S.A. 85:8027-8031 (1988); Ulmer et al., Science259:1745-1748 (1993)). The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Felgner and Ringold,Science 337:387-388 (1989)). Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. The use of lipofection to introduce exogenous genesinto the specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly advantageous in a tissue with cellularheterogeneity, for example, pancreas, liver, kidney, and the brain.Lipids may be chemically coupled to other molecules for the purpose oftargeting (see Mackey, et. al., supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins for example, antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

[0164] Other molecules are also useful for facilitating transfection ofa nucleic acid in vivo, for example, a cationic oligopeptide (e.g.,International Patent Publication WO95/21931), peptides derived from DNAbinding proteins (e.g., International Patent Publication WO96/25508), ora cationic polymer (e.g., International Patent Publication WO95/21931).

Naked DNA Delivery Systems

[0165] It is also possible to introduce the vector in vivo as a nakedDNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Naked DNA vectors for gene therapy can be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)).

[0166] Uses for NFIF Polvpeptides

[0167] As mentioned above, in certain situations it is desirable toinduce NFκB in order to initiate or increase the extent of an immuneresponse. In order to induce NFκB, the NFIF polypeptides of the presentinvention can be introduced into the body of a patient by a variety ofmethods.

[0168] The methods used to introduce NFIF into a patient's body includedirect administration of purified NFIF polypeptides or introduction ofnucleic acid encoding NFIF polypeptides in expression vectors whichexpress the polypeptides in the patient's body.

[0169] In embodiments involving direct administration of NFIFpolypeptides, the NFIF polypeptides are prepared using the host cellexpression systems such as those described above. The polypeptides arepurified using conventional purification methods and then combined witha suitable biologically-compatible solution as described in detailbelow. The solution containing the polypeptide is introduced into thepatient by topical, oral, parenteral, intranasal, subcutaneous orintraoccular routes. Once in the body, the polypeptide is able to exertits effect, inducing NFκB and thereby resulting in an increase in theactivity of NFκB-regulated pathways, including immune responses.

[0170] In embodiments involving administration of nucleic acids encodingNFIF polypeptides, viral or non-viral systems utilizing expressionvectors capable of expressing NFIF polypeptides can be introduced intothe patient's body. Any of the viral or non-viral transfection systemsdescribed above may be used. The viral and non-viral vector systems arealso combined with an appropriate biologically-compatible solution tofacilitate their introduction into the body. Once a viral or non-viralvector is introduced into the body, the NFIF polypeptide encodingnucleic acid may integrate into the host's genome, providing stable,long-term expression of NFIF polypeptides which exert their effect asdescribed above. Transient expression may be provided by systems thatintroduce the nucleic acid into cells, but do not integrate into thegenome.

[0171] The NFIF-14b and NFIF-7a polypeptides contain signal sequences,indicating these polypeptides are capable of being expressed in one cellwhile exerting their effect in another cell. Accordingly, the NFIFpolypeptides may be expressed by cells that then release NFIFpolypeptide into the bloodstream or other transport system (lymph, etc.)where they are transported to tissues where they exert their effect onNFκB induction.

[0172] The discussion which follows describes how to identifycompositions and methods for regulating NFκB induction based on thepresent discovery of the NFIF polypeptides.

[0173] Compositions

[0174] The present invention provides compositions in a biologicallycompatible (biocompatible) solution comprising the polypeptides, nucleicacids, and vectors of the invention. A biologically compatible solutionis a solution in which the polypeptide, nucleic acid or vector of theinvention is maintained in an active form, e.g., in a form able toeffect a biological activity. For example, a polypeptide of theinvention could have NFκB activation or deactivation activity; anantibody (which is itself a polypeptide) would bind a polypeptide of theinvention; a nucleic acid would be able to replicate, translate amessage, or hybridize to a complementary nucleic acid; and a vectorwould be able to transfect a target cell. Generally, such a biologicallycompatible solution will be an aqueous buffer, e.g., Tris, phosphate, orHEPES buffer, containing salt ions. Usually the concentration of saltions will be similar to physiological levels. In a specific embodiment,the biocompatible solution is a pharmaceutically acceptable composition.Biologically compatible solutions may include stabilizing agents andpreservatives.

[0175] Such compositions can be formulated for administration bytopical, oral, parenteral, intranasal, subcutaneous, and intraocular,routes. Parenteral administration is meant to include intravenousinjection, intramuscular injection, intraarterial injection or infusiontechniques. The composition may be administered parenterally in dosageunit formulations containing standard, well known nontoxicphysiologically acceptable carriers, adjuvants and vehicles as desired.

[0176] The preferred sterile injectable preparations can be a solutionor suspension in a nontoxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (e.g. monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride, or mixtures of such salts),Ringer's solution, dextrose, water, sterile water, glycerol, ethanol,and combinations thereof. 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media. Any bland fixedoil can be employed including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

[0177] The composition medium can also be a hydrogel which is preparedfrom any biocompatible or non-cytotoxic (homo or hetero) polymer, suchas a hydrophilic polyacrylic acid polymer that can act as a drugabsorbing sponge. Such polymers have been described, for example, inapplication WO93/08845, the entire contents of which are herebyincorporated by reference. Certain of them, such as, in particular,those obtained from ethylene and/or propylene oxide are commerciallyavailable. A hydrogel can be deposited directly onto the surface of thetissue to be treated, for example, during surgical intervention.

[0178] Another preferred embodiment of the present invention relates toa pharmaceutical composition comprising a replication defectiverecombinant virus and poloxamer. More specifically, the inventionrelates to a composition comprising a replication defective recombinantvirus comprising a nucleic acid encoding an NFIF polypeptide andpoloxamer. A preferred poloxamer is Poloxamer 407, which is commerciallyavailable (BASF, Parsippany, N.J.) and is a non-toxic, biocompatiblepolyol, and is most preferred. A poloxamer impregnated with recombinantviruses may be deposited directly on the surface of the tissue to betreated, for example during a surgical intervention. Poloxamer possessesessentially the same advantages as a hydrogel while having a lowerviscosity.

[0179] The present invention also relates to compositions useful in themanufacture of medicaments intended for the treatment of subjectsaffected with an NFκB-regulated inflammatory response and compositionsuseful in the manufacture of medicaments intended to prevent subjectsfrom being affected with an NFκB-regulated inflammatory response.Accordingly, the present invention includes the use of an NFIFpolypeptide according to the invention for the manufacture of amedicament intended for the treatment and/or prevention of anNFκB-regulated inflammatory response. The present invention alsoincludes the use of a nucleic acid according to the invention, encodingan NFIF polypeptide, for the manufacture of a medicament intended forthe treatment and/or prevention of an NFκB-regulated inflammatoryresponse. The present invention further includes the use of arecombinant vector according to the invention, comprising a nucleic acidencoding an NFIF polypeptide, for the manufacture of a medicamentintended for the treatment and/or prevention of an NFκB-regulatedinflammatory response. The present invention also includes the use of adefective recombinant viral vector according to the invention for themanufacture of a medicament intended for the treatment and/or preventionof an NFκB-regulated inflammatory response.

[0180] Another aspect of the present invention relates to the use ofcompositions comprising cells genetically modified e vivo with arecombinant virus and compositions comprising cells containing suchrecombinant viruses which are implanted in the body, facilitatingprolonged and effective expression in vivo of an NFIF polypeptideaccording to the invention.

[0181] Therapeutic Compounds Based on NFIF that Inhibit Activation ofNFκB

[0182] As explained above, in certain situations, it is desirable toreduce or inhibit NFκB-regulated immune responses. By inhibiting theexpression of the NFIF gene or interfering with the activity of NFIFpolypeptides, it is possible to inhibit induction of NFκB andconsequently inhibit or prevent NFκB-regulated immune responses thatresult in atherosclerosis and other diseases.

[0183] There are a variety of therapeutic compounds which may be used toinhibit NFIF expression or activity. These therapeutic compounds may benucleic acids, polypeptides, peptides or non-peptide small molecules. Inone embodiment, antisense nucleic acids are used to decrease expressionof the NFIF gene by inhibiting processing (splicing) of the NFIF primarytranscript. In another embodiment, ribozymes that cleave NFIF mRNA areused, preventing the synthesis of NFIF. Polypeptides of the presentinvention include antibodies or other binding proteins that bind NFIFpolypeptides and interfere with their ability to activate NFκBinduction. In addition, compounds such as small molecule inhibitorswhich inhibit NFIF gene expression or the activity of NFIF polypeptidescan be identified in reporter gene assays as described above andadministered to patients to inhibit NFIF expression/activity. The samemethods described above for introducing NFIF polypeptides and nucleicacids encoding NFIF polypeptides into a patient's body are used toadminister the various anti-NFIF compositions.

[0184] Antisense Nucleic Acids The down regulation of gene expressionusing antisense nucleic acids can be achieved at the translational ortranscriptional level. Antisense nucleic acids of the invention arepreferably nucleic acid fragments capable of specifically hybridizingwith all or part of a nucleic acid encoding NFIF or the correspondingmessenger RNA. In addition, antisense nucleic acids may be designed oridentified which decrease expression of the NFIF gene by inhibitingsplicing of its primary transcript. With knowledge of the structure andpartial sequence of the NFIF gene, such antisense nucleic acids can bedesigned and tested for efficacy.

[0185] The antisense nucleic acids are preferably oligonucleotides andmay consist entirely of deoxyribo-nucleotides, modifieddeoxyribonucleotides, or some combination of both. The antisense nucleicacids can be synthetic oligonucleotides. The oligonucleotides may bechemically modified, if desired, to improve stability and/orselectivity. Since oligonucleotides are susceptible to degradation byintracellular nucleases, the modifications can include, for example, theuse of a sulfur group to replace the free oxygen of the phosphodiesterbond. This modification is called a phosphorothioate linkage.Phosphorothioate antisense oligonucleotides are water soluble,polyanionic, and resistant to endogenous nucleases. In addition, when aphosphorothioate antisense oligonucleotide hybridizes to its targetsite, the RNA-DNA duplex activates the endogenous enzyme ribonuclease(Rnase) H, which cleaves the mRNA component of the hybrid molecule.

[0186] In addition, antisense oligonucleotides with phosphoramidite andpolyamide (peptide) linkages can be synthesized. These molecules shouldbe very resistant to nuclease degradation. Furthermore, chemical groupscan be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5)of pyrimidines to enhance stability and facilitate the binding of theantisense oligonucleotide to its target site. Modifications may include2′ deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxyphosphorothioates, modified bases, as well as other modifications knownto those of skill in the art.

[0187] The antisense nucleic acids can also be DNA sequences whoseexpression in the cell produces RNA complementary to all or part of theNFIF mRNA. Antisense nucleic acids can be prepared by expression of allor part of a sequence selected from the group consisting of the sequencein FIG. 3, in the opposite orientation, as described in EP 140308. Anylength of antisense sequence is suitable for practice of the inventionso long as it is capable of down-regulating or blocking expression ofNFIF. Preferably, the antisense sequence is at least 20 nucleotides inlength. The preparation and use of antisense nucleic acids, DNA encodingantisense RNAs and the use of oligo and genetic antisense is disclosedin WO92/15680, the contents of which are incorporated herein byreference.

[0188] One approach to determining the optimum fragment of NFIF to usein an antisense nucleic acid treatment method involves preparing randomfragments of NFIF cDNA by mechanical shearing, enzymatic treatment, andcloning the fragment into any of the vector systems described herein.Individual clones or pools of clones are used to infect NFIF-expressingcells, and effective antisense NFIF cDNA fragments are identified bymonitoring NFIF expression at the RNA or protein level.

[0189] The retroviral, adeno-associated viral, and adenoviral vectorsystems discussed hereinabove may all be used to introduce and expressantisense nucleic acids in cells. Antisense synthetic oligonucleotidesmay be introduced in a variety of ways, including the methods discussedhereinbelow.

[0190] Ribozymes

[0191] Reductions in the levels of NFIF polypeptide may be accomplishedusing ribozymes. Ribozymes are catalytic RNA molecules (RNA enzymes)that have separate catalytic and substrate binding domains. Thesubstrate binding sequence combines by nucleotide complementarity and,possibly, nonhydrogen bond interactions with its target sequence. Thecatalytic portion cleaves the target RNA at a specific site. Thesubstrate domain of a ribozyme can be engineered to direct it to aspecified mRNA sequence. The ribozyme recognizes and then binds a targetmRNA through complementary base-pairing. Once it is bound to the correcttarget site, the ribozyme acts enzymatically to cut the target mRNA.Cleavage of the NFIF mRNA by a ribozyme destroys its ability to directsynthesis of NFIF polypeptide. Once the ribozyme has cleaved its targetsequence, it is released and can repeatedly bind and cleave at otherNFIF mRNAs.

[0192] Examples of ribozymes for use in the practice of the presentinvention include a variety of motifs, for example, hammerhead motif,hairpin motif, a hepatitis delta virus, group I intron or RnaseP RNA (inassociation with an RNA guide sequence) motif or Neurospora VS RNAmotif. Hammerhead motifs are described by Rossi et al., 1992,AidsResearch and Human Retroviruses, 8, 183. Hairpin motifs are described inHampel and Tritz, 1989, Biochemistry, 28, 4929, and Hampel et al., 1990,Nucleic Acids Res., 18, 299. The hepatitis delta virus motif isdescribed by Perrotta and Been, 1992, Biochemistry, 31, 16, the RnasePmotif is described by Guerrier-Takada et al., 1983, Cell, 35, 849, theNeurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, 1990, Cell, 61, 685-696; Saville and Collins, 1991, Proc. Natl.Acad. Sci. USA, 88, 8826-8830; Collins and Olive, 1993, Biochemistry,32, 2795-2799) the Group I intron motif is described by Cech et al.,U.S. Pat. No. 4,987,071.

[0193] One approach in preparing a ribozyme is to synthesize chemicallyan oligodeoxyribonucleotide with a ribozyme catalytic domain (˜20nucleotides) flanked by sequences that hybridize to the target NFIF mRNAafter transcription. The oligodeoxyribonucleotide is amplified by usingthe substrate binding sequences as primers. The amplification product iscloned into a eukaryotic expression vector.

[0194] Ribozymes possessing a hammerhead or hairpin structure areprepared readily since these catalytic RNA molecules can be expressedwithin cells from eukaryotic promoters (e.g., Scanlon et al., 1991,Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992,Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66,1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al.,1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992,Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990, Science, 247,1222-1225)). A ribozyme of the present invention can be expressed ineukaryotic cells from the appropriate DNA vector. If desired, theactivity of the ribozyme may be augmented by its release from theprimary transcript by a second ribozyme (Ohkawa et al., 1992, NucleicAcids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19,5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55).

[0195] In one approach to preparing ribozymes, ribozymes are expressedfrom transcription units inserted into DNA, RNA, or viral vectors.Transcription of the ribozyme sequences are driven from a promoter foreukaryotic RNA polymerase I (pol (I), RNA polymerase II (pol II), or RNApolymerase III (pol III). Transcripts from pol II or pol III promoterswill be expressed at high levels in all cells; the levels of a given polII promoter in a given cell type will depend on nearby gene regulatorysequences. Prokaryotic RNA polymerase promoters are also used, providingthat the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells (Elroy-Stein and Moss, 1990, Proc. Nati. Acad. Sci.USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72;Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990,Mol. Cell. Biol., 10, 4529-37). It has been demonstrated that ribozymesexpressed from these promoters can function in mammalian cells(Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang etal., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci.USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 8000-4).

[0196] In one embodiment of the present invention, a transcription unitexpressing a ribozyme that cleaves NFIF RNA is inserted into a plasmidDNA vector, a retrovirus vector, an adenovirus DNA viral vector or anadeno-associated virus vector. The recombinant vectors are preferablyDNA plasmids or adenovirus vectors. However, other mammalian cellvectors that direct the expression of RNA may be used for this purpose.The vectors are delivered as recombinant viral particles. DNA may bedelivered alone or complexed with various vehicles. The DNA, DNA/vehiclecomplexes, or the recombinant virus particles are locally administeredto the site of treatment, as discussed below. Preferably, recombinantvectors capable of expressing the ribozymes are locally delivered asdescribed below, and persist in target cells. Once expressed, theribozymes cleave the target NFIF mRNA.

[0197] Ribozymes may be administered to a patient by a variety ofmethods. They may be added directly to target tissues, complexed withcationic lipids, packaged within liposomes, or delivered to target cellsby other methods known in the art. Localized administration to thedesired tissues may be done by catheter, infusion pump or stent, with orwithout incorporation of the ribozyme in biopolymers as discussedhereinbelow. Alternative routes of delivery include, but are not limitedto, intravenous injection, intramuscular injection, subcutaneousinjection, aerosol inhalation, oral (tablet or pill form), topical,systemic, ocular, intraperitoneal and/or intrathecal delivery. Moredetailed descriptions of ribozyme delivery and administration areprovided in Sullivan et al., PCT WO94/02595 and Draper et al., PCTWO93/23569, which are incorporated by reference herein.

[0198] Antibodies

[0199] The present invention provides antibodies against the NFIFpolypeptide. These antibodies may be monoclonal antibodies or polyclonalantibodies. The present invention includes chimeric, single chain, andhumanized antibodies, as well as Fab fragments and the products of anFab expression library, and Fv fragments and the products of an Fvexpression library.

[0200] Polyclonal antibodies may be prepared against an antigenicfragment of an NFIF polypeptide. Antibodies may also be generatedagainst the intact NFIF protein or polypeptide, or against a fragment,derivative, or epitope of the protein or polypeptide. Antibodies may beobtained following the administration of the protein, polypeptide,fragment, derivative, or epitope to an animal, using the techniques andprocedures known in the art.

[0201] Monoclonal antibodies may be prepared using the method ofMishell, B. B., et al., Selected Methods In Cellular Immunology, (W. H.Freeman, ed.) San Francisco (1980). Briefly, a polypeptide of thepresent invention is used to immunize spleen cells of Balb/C mice. Theimmunized spleen cells are fused with myeloma cells. Fused cellscontaining spleen and myeloma cell characteristics are isolated bygrowth in HAT medium, a medium which kills both parental cells, butallows the fused products to survive and grow.

[0202] The monoclonal antibodies of the present invention may be“humanized” to prevent the host from mounting an immune response to theantibodies. A “humanized antibody” is one in which the complementaritydetermining regions (CDRs) and/or other portions of the light and/orheavy variable domain framework are derived from a non-humanimmunoglobulin, but the remaining portions of the molecule are derivedfrom one or more human immunoglobulins. Humanized antibodies alsoinclude antibodies characterized by a humanized heavy chain associatedwith a donor or acceptor unmodified light chain or a chimeric lightchain, or vice versa. The humanization of antibodies may be accomplishedby methods known in the art (see, e.g. G. E. Mark and E. A. Padlan,“Chapter 4. Humanization of Monoclonal Antibodies”, The Handbook ofExperimental Pharmacology Vol. 113, Springer-Verlag, New York, 1994).Transgenic animals may be used to express humanized antibodies.

[0203] Techniques known in the art for the production of single chainantibodies can be adapted to produce single chain antibodies to theimmunogenic polypeptides and proteins of the present invention.

[0204] In a preferred embodiment, an anti-NFIF antibody is used to bindto and inhibit the activity of NFIF in a patient.

[0205] The anti-NFIF antibodies are also useful in assays for detectingor quantitating levels of NFIF. In one embodiment, these assays providea clinical diagnosis and assessment of NFIF in various disease statesand a method for monitoring treatment efficacy. An example of ananti-NFIF antibody used to bind NFIF and identify its presence intissues is provided in Example 5. These anti-NFIF antibodies mayadditionally be used to quantitate NFIF in a tissue sample.

[0206] Methods of Treatment

[0207] The present invention provides methods of treatment whichcomprise the administration to a human or other animal of an effectiveamount of a composition of the invention.

[0208] Effective amounts may vary, depending on the age, type andseverity of the condition to be treated, body weight, desired durationof treatment, method of administration, and other parameters. Effectiveamounts are determined by a physician or other qualified medicalprofessional. In most cases, the dosage levels may be adjusted so thatthe desired levels of NFIF or other therapeutic compounds can beachieved and maintained.

[0209] Polypeptides according to the invention are generallyadministered in doses of about 0.01 mg/kg to about 100 mg/kg, morepreferably about 0.1 mg/kg to about 50 mg/kg, and even more preferably.about 1 mg/kg to about 10 mg/kg of body weight per day.

[0210] Neutralizing antibodies according to the invention may bedelivered as a bolus only, infused over time or both administered as abolus and infused over time. Although the dosage amount will vary basedon the parameters above, and on the binding ability of the antibody, adose 0.2 to 0.6 mg/kg may be given as a bolus followed by a 2 to 12 hourinfusion period. Alternatively, multiple bolus injections areadministered every other day or every third or fourth day as needed.Dosage levels may be adjusted as determined by NFIF levels and/or NFκBinduction levels.

[0211] As discussed hereinabove, recombinant viruses may be used tointroduce both DNA encoding NFIF and subfragments of NFIF as well asantisense nucleic acids. Recombinant viruses according to the inventionare generally formulated and administered in the form of doses ofbetween about 10⁴ and about 10¹⁴ pfu. In the case of AAVs andadenoviruses, doses of from about 10⁶ to about 10¹¹ pfu are preferablyused. The term pfu (“plaque-forming unit”) corresponds to the infectivepower of a suspension of virions and is determined by infecting anappropriate cell culture and measuring the number of plaques formed. Thetechniques for determining the pfu titre of a viral solution are welldocumented in the prior art.

[0212] Ribozymes according to the present invention may be administeredin amounts ranging from about 5 to about 50 mg/kg/day in apharmaceutically acceptable carrier. Dosage levels may be adjusted basedon the measured therapeutic efficacy.

[0213] Appropriate levels of inhibitor or enhancer molecules may bedetermined by qualified medical personnel using the parameters discussedabove.

[0214] Methods for Increasing the Level of NFIF Polypeptide Activity

[0215] The methods for increasing the expression or activity of NFIFpolypeptide include, but are not limited to, administration of acomposition comprising the NFIF polypeptide, administration of acomposition comprising an expression vector which encodes the NFIFpolypeptide, administration of a composition comprising an enhancermolecule which enhances the activity of the NFIF polypeptide andadministration of an enhancer molecule which increases expression of theNFIF gene.

[0216] Methods Utilizing NFIF Polypeptides

[0217] In one embodiment, the level of NFIF activity is increasedthrough the administration of a composition comprising the NFIFpolypeptide. This composition may be administered in a convenientmanner, such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, or intradermal routes. Thecomposition may be administered directly or it may be encapsulated (e.g.in a lipid system, in amino acid microspheres, or in globulardendrimers). The polypeptide may, in some cases, be attached to anotherpolymer such as serum albumin or polyvinyl pyrrolidone.

[0218] Methods Utilizing Vectors that Express NFIF

[0219] In another embodiment, the level of NFIF is increased through theuse of gene therapy, that is, through the administration of compositioncomprising a nucleic acid which encodes and directs the expression ofthe NFIF polypeptide. In this embodiment, the NFIF polypeptide is clonedinto an appropriate expression vector. Possible vector systems andpromoters are extensively discussed above. The expression vector istransferred into the target tissue using one of the vector deliverysystems discussed above. This transfer is carried out either ex vivo ina procedure in which the nucleic acid is transferred to cells in thelaboratory and the modified cells are then administered to the human orother animal, or in vivo in a procedure in which the nucleic acid istransferred directly to cells within the human or other animal. Inpreferred embodiments, an adenoviral vector system is used to deliverthe expression vector. If desired, a tissue specific promoter isutilized in the expression vector as described above.

[0220] Non-viral vectors may be transferred into cells using any of themethods known in the art, including calcium phosphate coprecipitation,lipofection (synthetic anionic and cationic liposomes),receptor-mediated gene delivery, naked DNA injection, electroporationand bioballistic or particle acceleration.

[0221] Methods Utilizing an Enhancer Molecule which Enhances theActivity of NFIF

[0222] In another embodiment, the activity of NFIF is enhanced byenhancer molecules that increase the activity of NFIF or increase itsappropriate recognition by cellular binding sites. These enhancermolecules may be introduced by the same methods discussed above for theadministration of polypeptides.

[0223] Methods Utilizing an Enhancer Molecule which Increases NFIF GeneExpression

[0224] In another embodiment, the level of NFIF is increased through theuse of small molecular weight compounds, which can upregulate NFIFexpression at the level of transcription, translation, orpost-translation. These compounds may be administered by the samemethods discussed above for the administration of polypeptides.

[0225] Methods for Treating or Preventing an NFκB-regulated InflammatoryResponse

[0226] The present invention includes methods for the treatment orprevention of NFκB-regulated inflammatory responses including, but notlimited to, rheumatoid arthritis, atherosclerosis, autoimmune diseases,viral diseases, NSAID-induced gastropathy, neurodegenerative diseases,scrapie, sepsis, apoptosis, Crohn's disease, renal disease, restenosis,brain injury/inflammation, Alzheimer's disease, asthma, and improperlyregulated expression of pleiotropic cytokines.

[0227] These methods include, but are not limited to, administration ofa composition comprising an antisense nucleic acid, administration of acomposition comprising an intracellular binding protein such as anantibody, administration of an inhibitory molecule which inhibits theactivity of NFIF, for example, a composition comprising an expressionvector encoding a subfragment of NFIF or a small molecular weightmolecule, including administration of a small molecular weight compoundwhich down regulates NFIF expression at the level of transcription,translation or post-translation, administration of a ribozyme whichcleaves mRNA encoding NFIF, administration of a medicament manufacturedusing an NFIF polypeptide, administration of a medicament manufacturedusing a nucleic acid encoding an NFIF polypeptide, administration of amedicament manufactured using a recombinant vector which includesnucleic acid encoding an NFIF polypeptide and administration of amedicament manufactured using a defective recombinant viral vector whichincludes nucleic acid encoding an NFIF polypeptide.

[0228] Methods for Lowering Levels of NFIF Polypeptide Activity

[0229] The methods for decreasing the expression of NFIF polypeptide inorder to decrease NFκB induction include, but are not limited to,administration of a composition comprising an antisense nucleic acid,administration of a composition comprising an intracellular bindingprotein such as an antibody, administration of an inhibitory moleculewhich inhibits the activity of NFIF, for example, a compositioncomprising an expression vector encoding a subfragment of NFIF or asmall molecular weight molecule, including administration of a smallmolecular weight compound which down regulates NFIF expression at thelevel of transcription, translation or post-translation, andadministration of a ribozyme which cleaves mRNA encoding NFIF.

[0230] Methods Utilizing Antisense Nucleic Acids

[0231] In one embodiment, a composition comprising an antisense nucleicacid is used to down-regulate or block the expression of NFIF. In onepreferred embodiment, the nucleic acid encodes antisense RNA molecules.In this embodiment, the nucleic acid is operably linked to signalsenabling expression of the nucleic acid sequence and is introduced intoa cell utilizing, preferably, recombinant vector constructs, which willexpress the antisense nucleic acid once the vector is introduced intothe cell. Examples of suitable vectors includes plasmids, adenoviruses,adeno-associated viruses, retroviruses, and herpes viruses. Preferably,the vector is an adenovirus. Most preferably, the vector is areplication defective adenovirus comprising a deletion in the E1 and/orE3 regions of the virus.

[0232] In another embodiment, the antisense nucleic acid is synthesizedand may be chemically modified to resist degradation by intracellularnucleases, as discussed above. Synthetic antisense oligonucleotides canbe introduced to a cell using liposomes. Cellular uptake occurs when anantisense oligonucleotide is encapsulated within a liposome. With aneffective delivery system, low, non-toxic concentrations of theantisense molecule can be used to inhibit translation of the targetmRNA. Moreover, liposomes that are conjugated with cell-specific bindingsites direct an antisense oligonucleotide to a particular tissue.

[0233] Methods Utilizing Neutralizing Antibodies and Other BindingProteins

[0234] In another embodiment, the expression of NFIF is down-regulatedor blocked by the expression of a nucleic acid sequence encoding anintracellular binding protein which is capable of selectivelyinteracting with NFIF. WO 94/29446 and WO 94/02610, the contents ofwhich are incorporated herein by reference, disclose cellulartransfection with genes encoding an intracellular binding protein. Anintracellular binding protein includes any protein capable ofselectively interacting, or binding, with NFIF in the cell in which itis expressed and of neutralizing the function of bound NFIF. Preferably,the intracellular binding protein is a neutralizing antibody or afragment of a neutralizing antibody. More preferably, the intracellularbinding protein is a single chain antibody.

[0235] WO 94/02610 discloses preparation of antibodies andidentification of the nucleic acid encoding a particular antibody. UsingNFIF or a fragment thereof, a specific monoclonal antibody is preparedby techniques known to those skilled in the art. A vector comprising thenucleic acid encoding an intracellular binding protein, or a portionthereof, and capable of expression in a host cell is subsequentlyprepared for use in the method of this invention.

[0236] Alternatively, NFIF activity can be blocked by administration ofa neutralizing antibody into the circulation. Such a neutralizingantibody can be administered directly as a protein, or it can beexpressed from a vector (with a secretory signal).

[0237] Methods Utilizing an Inhibitory Molecule which Prevents NFIF GeneExpression

[0238] In another embodiment, inhibitory molecules, including smallmolecular weight compounds, are able to down regulate NFIF expression atthe level of transcription, translation or post-translation. In order toidentify such inhibitory molecules, the reporter gene systems describedabove may be used. These inhibitory molecules may be combined with apharmaceutically acceptable carrier and administered using conventionalmethods known in the art.

[0239] Methods Utilizing Ribozymes

[0240] Ribozymes may be administered to cells by encapsulation inliposomes, by iontophoresis, by incorporation into hydrogels,cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheresor by any of a variety of other methods dicussed above. The ribozyme maybe delivered to a target tissue by direct injection or by use of acatheter, infusion pump or stent. Alternative routes of delivery includeintravenous injection, intramuscular injection, subcutaneous injection,aerosol inhalation, oral (tablet or pill form), topical, systemic,ocular, intraperitoneal and/or intrathecal delivery.

[0241] In preferred embodiments, a ribozyme-encoding sequence is clonedinto a DNA expression vector. Transcription of the ribozyme sequence isdriven from an eukaryotic RNA polymerase II (pol II), or RNA polymeraseIII (po lII) promoter. The expression vector can be incorporated into avariety of vectors including the viral DNA vectors such as adenovirus oradeno-associated virus vectors discussed above.

[0242] In a preferred embodiment of the invention, a transcription unitexpressing a ribozyme that cleaves NFIF RNA is inserted into anadenovirus DNA viral vector. The vector is delivered as recombinantviral particles and is locally administered to the site of treatment,through the use of a catheter, stent or infusion pump.

EXAMPLES Example 1

[0243] Method for Cloning NFIF-14b and NFIF-7a

[0244] To isolate the DNA encoding NFIF-14b and NFIF-7a, a cDNA librarywas prepared from human vascular smooth muscle cell (SMC) total RNAusing a Clontech Marathon™ cDNA library synthesis kit. The cDNAsynthesis kit also includes a control human placenta DNA library. Thelibraries are linear and amplifiable by PCR.

[0245] The NFIF-14b and NFIF-7a genes were obtained by PCR performed onboth Human Vascular SMC and Human Placenta cDNA marathon libraries.

[0246] To perform the PCR reactions, standard PCR components [PerkinElmer 10X buffer and Taq Gold Polymerase] were used and the reaction wasperformed as follows. The reaction mixtures were heated to 94° C. for 10minutes, then thermocycled 10 times with a denaturing step of 94° C. for30 seconds, an annealing step of 65° C. for 30 seconds, and an extensionstep of 72° C. for 1.5 minutes. Following these ten cycles, the reactionwas thermocycled 30 times with a denaturing step of 94° C. for 30seconds, an annealing step of 46° C. for 30 seconds., and an extensionstep of 72° C. for 1.5 minutes. Following these 30 cycles, the reactionswere incubated at 72° C. for 7 minutes and stored at 4° C. The PCRreactions were performed in a Perkin Elmer 9600 thermocycler.

[0247] The PCR product from each PCR was ligated into pCR2. 1(Invitrogen) and transformed in DH5α-E.coli. Clones were picked andgrown up in 5 ml of LB-amp; plasmids were then isolated and digestedwith EcoRI to check for the size of the released insert. Five out offifteen clones gave bands of size 1.2-1.3 Kb, suggestive of the targetgene and were sequenced.

[0248] Putative NFIF clones were identified based on an open readingframe and sequence alignment of the sequence against a predicted 5′sequence. Clones Nos. 14b and 7a were chosen for furthercharacterization.

[0249] Large plasmid preparations were grown up from original glycerolstocks of clone 7 and 14, and isolated using a Qiagen MaxiPrep Kit.

Example 2

[0250] Subcloning of Clone 7a and 14b into a Eukarvotic ExpressionVector

[0251] Clones 14b and 7a were further subcloned into the plasmid vectorpcDNA3. Imyc-his available from Invitrogen. This expression vectorincludes a strong promoter for high level expression in mammalian cellsand a selection marker for generating stable cell lines. A C-terminalfusion tag in the vector features a polyhistidine sequence for rapidpurification and a myc epitope for convenient detection with an anti-mycantibody.

[0252] PCR reactions were performed using PCR primers

[0253] 5 ′hasm-5′-tccaccatggcgctggtgcgcgcactc-3′ and

[0254] fushasm3′-3′-ctggatatcgtaattgtgctttatataaagctg-5′ and the pCR2.1construct for each full length clone as template. PCR conditions using475pg of pCR2.1 clone 7a or 500 pg of pCR2.1 clone 14b were as follows:the reaction mixtures were heated to 95° C. for 10 minutes, thenthermocycled 30 times with a denaturing step of 95° C. for 30 seconds,an annealing step of 52° C. for 30 seconds, and then an extension stepof 72° C. for 1.5 minutes. Following these 30 cycles, the reactions wereincubated at 72° C. for 7 minutes, and stored at 4° C.

[0255] The PCR product was. ligated into pCR2.1 and the insert DNAsequenced utilizing a Big Dye Terminator Cycle Sequencing Ready Reactionand a Perkin Elmer Applied Biosystems ABI Prism 377 DNA Sequencer.Sequencing demonstrated that the PCR primer removed the natural stopcodon (TAG) and introduced an EcoRV site.

[0256] The positive constructs of NFIF-14b and -7a were digested withEcoRI and EcoRV and subcloned into pcDNA3.1 mychis (Invitrogen) cut withEcoRI and EcoRV. This resulted in the myc tag being fused to the 3′ endof the NFIF cDNAs. These constructs were called pcDNA3.1mychasm7a andpcDNA3.1hasml4bmyc.

Example 3

[0257] Preparation of Deletion Variants of NFIF

[0258] Deletion mutants of NFIF-14b and NFIF-7a were prepared by PCRreactions using the pcDNA3.1mychasm7a and pcDNA3.1hasm 14bmyc plasmidsdescribed above. Two sets of PCR primers were used to prepare thedeletion mutants: hasm313mut + hasm3′ mut:5′ gctccaccatgatatggacaggggatag 3′ 5′ gccactgtgctggatatcgtaattaac 3′hasm396mut + hasm3′ mut: 5′ gctccaccatgacaaccaccatccagagtc 3′5′ gccactgtgctggatatcgtaattaac 3′

[0259] The hasm 313+ hasm3′ mut primer pair produced the identicalsequence of the full length clone 14b or 7a cDNAs but on an open readingframe initiated downstream at bp313 (ATG). (Numbering is from the ATG inthe full length clone.) The consensus Kozak sequence was included in theforward primer to optimize translation.

[0260] The hasm396+ hasm3′ mut primer pair produced the identicalsequence of the full length cDNAs but on an open reading frame initiateddownstream at bp 394 (ATG).

[0261] The PCR products were ligated into pCR2.1 and the insert DNAsequenced. Each deletion mutant was cut with EcoRI/EcoRV and subclonedinto pcDNA3.1 mychis cut with EcoRI and EcoRV. NFIF-14b deletion cloneswith the correct sequence were named pcDNAmychis 14-313 and peDNAmychis14-396. NFIF-7a deletion clones with the correct sequence were namedpcDNAmychis 7-313, and pcDNAmychis 7-396.

[0262] Analysis of the above clones using in vitrotranscription/translation incorporating [³⁵S methionine] into thereaction [Promega TnT Quick Coupled Transcription Translation kit#L1170] produced bands of the size expected(NFIF 14b=51 kDa; NFIF14-313=40 kDa; NFIF 14-396=37 kDa; NFIF 7a=41 kDa; NFIF 7-313=30 kDa;and NFIF 7-396=27 kDa) for shortened open reading frames. The facilityof translation was important to determine before assessing functionalactivity and determining which domains were important for activity.

Example 4

[0263] Method for Transfecting Cells with NFIF-14b or NFIF-7a to ProduceStable Cell Lines Containing Plasmids

[0264] To prepare stable cell lines containing NFIF-14b, NFIF-7a and thedeletion mutants, Falcon 6 well plates were seeded with 2×10⁵ HEK293 orCOS-7 cells, the finalized cDNA NFIF constructs pcDNAmychis7,pcDNAmychis7-313, pcDNAmychis7-396; pcDNAmychis14, pcDNAmychis14-313,pcDNAmychis14-396 (0.8 ug) along with the Stratagene pNFκB-Luc reportergene vector (0.1 ug) and Clontech EGFP vector (0.1 ug), were transfectedusing 6 ul Lipofectamine and 200 ul Optimem into the 6 well plates (with800 ul fresh Optimem/well). The transfection was incubated for 4 hr at37° C. and then the plates were fed with complete media (3ml).

[0265] At 24, 48 and 72 hours post transfection lysates were made using200 ul 1× Reporter Lysis Buffer solution (E397A-Promega). Lysates wereincubated for 20 minutes on ice, vortexed, then centifuged at 12,000 rpmfor 5 minutes The supernatant was used to assess luciferase activity perthe instructions in the Stratagene pNFκB-luc reporter gene kit.

[0266] To prepare stable cells lines, duplicates of the above 48 hrcultures were split at a 1:100 ratio into complete growth media alongwith G418 for selection of cells incorporating the plasmids in theirDNA. Single cell clones were isolated and transferred into 48 wellculture dishes for growout. Clones were chosen according to theiractivity in the NFκB luciferase reporter assay.

[0267] Luciferase activity was measured from the cell lysates of all thetransient infected cell lines. 20 μl of lysate was added to a 96 wellCostar serocluster (#3789) white round bottom plate. 100 μl ofLuciferase Reagent (Promega E1501) was added directly before reading thesample in a 1450 Microbeta Walko jet.

[0268] Analysis of the results from 24 hour, 48 hour, 72 hour timepointsidentified the 48 hour timepoint as the most optimal for assessingactivity. The positive control for the assay was a 24 hour tumornecrosis factor (α) (TNFα) stimulation of the cells one dayposttransfection with the pcDNA3.1mychis vector. In HEK293 cells, thefull-length pcDNAmychis 7, pcDNAmychis14 cDNAs transient transfectionsdemonstrated luminometer count per second at ˜3.1 fold and ˜2.1 foldrespectively, above vector only (FIG. 5) and in COS-7 cells (FIG. 6)full-length clone pcDNAmychis 7 gave a 2.4 fold signal and pcDNAmychis14 a 2.3 fold increase in luminometer count per second above vectoronly.

[0269] The deletion mutants (pcDNAmychis 7-313, pcDNAmychis 7-396,pcDNAmychis 14-313 and pcDNAmychis 14-396) showed reduced activity.Assays run on other days using the 48 hour timepoint gave variability inresponse ranging between a 2-5 fold increase for clone 7 (full-length)and 2-4 fold for clone 14.

Example 5

[0270] Identification of Tissues Expressing or Containing NFIF Proteins

[0271] In order to determine which human tissues express the NFIFproteins, a Clontech pre-made Northern Blot (Human 12 lane #7780-1) wasprobed with randomly primed p32 labelled probes prepared from NFIF. Theresults can be seen in FIG. 8. As FIG. 8 illustrates, expression of NFIFis particularly evident in skeletal muscle, kidney, liver and placentaltissue.

[0272] In order to determine if the NFIF protein was associated withpathologies including atherosclerosis that involve inflammation and toidentify tissues that may be treated using the methods of the presentinvention, an immunocytochemical study was performed using a rabbitmonoclonal antibody designated 99-06 directed against a peptide antigen(SKGANASNPGPFGDV) derived from residues 65 to 79 of the NFIF protein.The peptide was synthesized at the 0.25 mmole scale using a solid phasemethodology FMOC (9-fluorenylmethyloxycarbonyl) protection scheme inconjunction with the HOBT/HBTU activation chemistry (Fields et al.,Peptide Research, 4:95-101 (1991)). An Applied Biosystems 433 PeptideSynthesizer running Applied Biosystems Fast-Moc coupling cycle was usedfor the synthesis of the peptide.

[0273] The peptide was cleaved for 1.5 hours at room temperature using acleavage reagent of 82.5% trifluoroacetic acid (TFA), 5% phenol, 5% H20,5% thioanisole, and 2.5% ethanedithiol (King et al., Intl. J. Peptideand Protein Research, 36, 255-266 (1990)). Following cleavage, thepeptide was precipitated with tert-butyl ether, washed, then dried for 1hour under vacuum. The peptide was then solubilized in 0.1% TFA/waterand purified using C18 reverse phase HPLC. A purity level of >95% wasachieved for the peptide, along with correct MALDI-TOF molecular weightdata. 10-30 mg of purified peptide was conjugated to keyhole limpethemocyanin using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).The preparation was dialyzed against PBS at 4° C. exhaustively. Thehapten/carrier preparation was mixed 1:1 with Freunds Complete adjuvantand used to inject rabbits for the production of antisera against thepeptide. The 15-residue peptide was used to prepare anti-NFIF antibodiesin rabbits using standard methods known in the art. Rabbits wereimmunized by subcutaneous injection with an emulsion of antigen/completeFreund's adjuvant (50% antigen solution/50% complete Freund's adjuvant).Complete Freund's adjuvant (CFA) was used for the initial immunizationbecause this adjuvant has been used very successfully in producingantibody responses to a diverse range of protein and peptide antigensand the incidence of adverse reactions is minimal to none. The injectionschedule described below has been optimized for use with Freund'sadjuvant. The area to be injected was shaved in order to permit visualassessment of any reaction to the emulsion. Prior to injection, theinjection sites were cleansed with diluted Nolvasan scrub solution.Injection sites were on the back starting between the shoulder blades.The injection sites were widely separated and kept close to the midlineto reduce the possibility of the animal scratching at the injectionsites. Typically, 100 μl containing 20 μg of antigen were injected in 5subcutaneous sites per rabbit for a total of 100 μg/rabbit. Subsequent,booster injections were given at 4 week intervals with an emulsion ofantigen/incomplete Freund's adjuvant. After six weeks, 5-30 mls of bloodwas collected to determine the titer of the antibody. If the antibodytiter was not sufficient, additional booster injections were given atweek intervals and blood samples collected every 2-4 weeks. Prior toeach antigen injection, the condition of the previous injection siteswere evaluated for adverse reactions. Animals giving a positive antibodyresponse were maintained and boosted and bled periodically in order togenerate a large stock of antisera.

[0274] Antibody titration experiments were conducted with Antibody 99-06to establish concentrations that yield minimal background and maximumdetection of signal. A serial dilution study demonstrated the highestsignal-to-noise ratio at a dilution of 1:500 and 1:1,000, and slidesincubated with these concentrations of antibody were analyzed by thepathologists. Antibody 99-06 was used as the primary antibody, and theprincipal detection system consisted of a Vector ABC-AP Kit (AK5002)with a Vector Red substrate kit that produced a fuschia-colored reddeposit (SK-5100). Tissues were also stained with a positive controlantibody (CD31) directed against the human leukocyte antigen (Stockingeret al., J. Immunol, 145(11):3889-97 (1990)) to ensure that the tissueantigens were preserved and accessible for immunocytochemical analysis.In addition to coronary artery samples, additional tissues that werestained and imaged during Phase II of this study were normal spleen,thymus, and lymph node. Staining was performed in the spleen, thymus andlymph node in order to assist in the identification of inflammatorysubsets of cells that expressed this protein.

[0275] Within the spleen, thymus, and lymph node, Antibody 99-06 showedstrong positive staining within the lymphocytes of the periarteriallymphatic sheath, the cords of Billroth, cortical thymic lymphocytes,germinal center cells in the lymph node, and within vascular endotheliumand smooth muscle. Other cell types that stained moderately to stronglypositive with this antibody included Schwann cells and cardiac myocytes.The signal was predominantly nuclear in the cell types examined, exceptfor cytoplasmic granularity that was seen within smooth muscle cells,and in macrophages that were filled with cellular debris in regions ofplaque or organizing thrombus.

[0276] Within normal coronary arteries, the antibody was largelynegative in the endothelium of the tunica intima except for occasionalcells showing a nuclear signal, and myointimal smooth muscle cellsshowed faint nuclear signals. Within the tunica media the antibody wasmoderately uniformly positive within smooth muscle cells. In theadventitia, the endothelium, vascular smooth muscle and inflammatorycells were moderately positive for staining. The internal and externalelastic lamellae were negative for staining.

[0277] Within the minimal atherosclerosis samples, the endotheliumshowed only occasional faint signals even in that overlying theatheroma. Within the superficial atheroma, the myointimal smooth musclecells showed only faint signals. In deeper regions of the atheroma,proliferating fibroblastic forms and foamy macrophages showed anincreased level of staining. In some samples that showed plaquescontaining cholesterol and areas of calcification, the smooth musclecells and foamy histiocytes surrounding the cholesterol-laden plaquesand calcified material showed increased levels of staining.

[0278] In the samples of moderate atherosclerosis, the endothelium, bothin the tunica intima and in areas of neovascularization, showedincreased staining with Antibody 99-06.

[0279] Inflammatory cells marginating along or subjacent to theendothelium were strongly positive for staining. Endothelium lining newvessels was moderately to strongly positive for staining, and in areas,the subendothelial myofibroblasts were less positive than associatedendothelium. In general, fibroblastic forms of myointimal cells weremuch less positive than either proliferating or histiocytic forms ofsmooth muscle cells. In addition, macrophages associated withcholesterol, calcification, or foamy macrophages within plaques showedan increased level of staining. The smooth muscle of the tunica mediawas fairly uniformly moderately positive within all samples in which themedia was intact, and smooth muscle within adventitial vessels wassimilarly positive.

[0280] In cases of severe atherosclerosis, much of the endotheliumlining the tunica intima was either denuded or the residual endotheliumwas faintly to moderatley positive for staining. The plaque-associatedinflammatory infiltrate showed very strong positive staining withinmacrophages and lymphocytes, including within regions ofneovascularization. Increased staining was also seen within macrophagesand lymphocytes adjacent to or surrounding calcifications andcholesterol and within areas of organizing thrombus. The endotheliumlining new vessels was strongly positive in these areas. Increasedsignal intensity was again seen within the histiocytic and proliferatingforms compared to fibroblastic forms of smooth muscle cells. In thesecases, inflammatory cells such as a subset of lymphocytes, plasma cells,and macrophages within the adventitia were also strongly positive forstaining.

[0281] In late stage areas of severe atherosclerosis with minimalinflammation and quiescent plaques, there was much less staining. Inthese samples, the endothelium was less intensely positive for staining,although residual lymphocytes and macrophages surrounding calcifiedmaterial showed positive staining.

[0282] Biological Material Useful in Practicing the Invention

[0283] A deposit of Human NFκB inducing factor 7a (NFIF-7a) cloned froma human vascular smooth muscle cell cDNA library subcloned into a pCR2.1plasmid vector and a deposit of Human NFκB inducing factor 14b(NFIF-14b) cloned from a human vascular smooth muscle cell cDNA librarysubcloned into a pCR2.1 plasmid vector will be made with the AmericanType Culture Collection. Access to these cultures will be availableduring pendency of the patent application to one determined by theCommissioner to be entitled thereto under 35 U.S.C. §122 and 37 C.F.R.§1.14.

1 5 1 453 PRT Homo sapiens 1 Met Ala Leu Val Arg Ala Leu Val Cys Cys LeuLeu Thr Ala Trp His 1 5 10 15 Cys Arg Ser Gly Leu Gly Leu Pro Val AlaPro Ala Gly Gly Arg Asn 20 25 30 Pro Pro Pro Ala Ile Gly Gln Phe Trp HisVal Thr Asp Leu His Leu 35 40 45 Asp Pro Thr Tyr His Ile Thr Asp Asp HisThr Lys Val Cys Ala Ser 50 55 60 Ser Lys Gly Ala Asn Ala Ser Asn Pro GlyPro Phe Gly Asp Val Leu 65 70 75 80 Cys Asp Ser Pro Tyr Gln Leu Ile LeuSer Ala Phe Asp Phe Ile Lys 85 90 95 Asn Ser Gly Gln Glu Ala Ser Phe MetIle Trp Thr Gly Asp Ser Pro 100 105 110 Pro His Val Pro Val Pro Glu LeuSer Thr Asp Thr Val Ile Asn Val 115 120 125 Ile Thr Asn Met Thr Thr ThrIle Gln Ser Leu Phe Pro Asn Leu Gln 130 135 140 Val Phe Pro Ala Leu GlyAsn His Asp Tyr Trp Pro Gln Asp Gln Leu 145 150 155 160 Ser Val Val ThrSer Lys Val Tyr Asn Ala Val Ala Asn Leu Trp Lys 165 170 175 Pro Trp LeuAsp Glu Glu Ala Ile Ser Thr Leu Arg Lys Gly Gly Phe 180 185 190 Tyr SerGln Lys Val Thr Thr Asn Pro Asn Leu Arg Ile Ile Ser Leu 195 200 205 AsnThr Asn Leu Tyr Tyr Gly Pro Asn Ile Met Thr Leu Asn Lys Thr 210 215 220Asp Pro Ala Asn Gln Phe Glu Trp Leu Glu Ser Thr Leu Asn Asn Ser 225 230235 240 Gln Gln Asn Lys Glu Lys Val Tyr Ile Ile Ala His Val Pro Val Gly245 250 255 Tyr Leu Pro Ser Ser Gln Asn Ile Thr Ala Met Arg Glu Tyr TyrAsn 260 265 270 Glu Lys Leu Ile Asp Ile Phe Gln Lys Tyr Ser Asp Val IleAla Gly 275 280 285 Gln Phe Tyr Gly His Thr His Arg Asp Ser Ile Met ValLeu Ser Asp 290 295 300 Lys Lys Gly Ser Pro Val Asn Ser Leu Phe Val AlaPro Ala Val Thr 305 310 315 320 Pro Val Lys Ser Val Leu Glu Lys Gln ThrAsn Asn Pro Gly Ile Arg 325 330 335 Leu Phe Gln Tyr Asp Pro Arg Asp TyrLys Leu Leu Asp Met Leu Gln 340 345 350 Tyr Tyr Leu Asn Leu Thr Glu AlaAsn Leu Lys Gly Glu Ser Ile Trp 355 360 365 Lys Leu Glu Tyr Ile Leu ThrGln Thr Tyr Asp Ile Glu Asp Leu Gln 370 375 380 Pro Glu Ser Leu Tyr GlyLeu Ala Lys Gln Phe Thr Ile Leu Asp Ser 385 390 395 400 Lys Gln Phe IleLys Tyr Tyr Asn Tyr Phe Phe Val Ser Tyr Asp Ser 405 410 415 Ser Val ThrCys Asp Lys Thr Cys Lys Ala Phe Gln Ile Cys Ala Ile 420 425 430 Met AsnLeu Asp Asn Ile Ser Tyr Ala Asp Cys Leu Lys Gln Leu Tyr 435 440 445 IleLys His Asn Tyr 450 2 364 PRT Homo sapiens 2 Met Ala Leu Val Arg Ala LeuVal Cys Cys Leu Leu Thr Ala Trp His 1 5 10 15 Cys Arg Ser Gly Leu GlyLeu Pro Val Ala Pro Ala Gly Gly Arg Asn 20 25 30 Pro Pro Pro Ala Ile GlyGln Phe Trp His Val Thr Asp Leu His Leu 35 40 45 Asp Pro Thr Tyr His IleThr Asp Asp His Thr Lys Val Cys Ala Ser 50 55 60 Ser Lys Gly Ala Asn AlaSer Asn Pro Gly Pro Phe Gly Asp Val Leu 65 70 75 80 Cys Asp Ser Pro TyrGln Leu Ile Leu Ser Ala Phe Asp Phe Ile Lys 85 90 95 Asn Ser Gly Gln GluAla Ser Phe Met Ile Trp Thr Gly Asp Ser Pro 100 105 110 Pro His Val ProVal Pro Glu Leu Ser Thr Asp Thr Val Ile Asn Val 115 120 125 Ile Thr AsnMet Thr Thr Thr Ile Gln Ser Leu Phe Pro Asn Leu Gln 130 135 140 Val PhePro Ala Leu Gly Asn His Asp Tyr Trp Pro Gln Val Tyr Ile 145 150 155 160Ile Ala His Val Pro Val Gly Tyr Leu Pro Ser Ser Gln Asn Ile Thr 165 170175 Ala Met Arg Glu Tyr Tyr Asn Glu Lys Leu Ile Asp Ile Phe Gln Lys 180185 190 Tyr Ser Asp Val Ile Ala Gly Gln Phe Tyr Gly His Thr His Arg Asp195 200 205 Ser Ile Met Val Leu Ser Asp Lys Lys Gly Ser Pro Val Asn SerLeu 210 215 220 Phe Val Ala Pro Ala Val Thr Pro Val Lys Ser Val Leu GluLys Gln 225 230 235 240 Thr Asn Asn Pro Gly Ile Arg Leu Phe Gln Tyr AspPro Arg Asp Tyr 245 250 255 Lys Leu Leu Asp Met Leu Gln Tyr Tyr Leu AsnLeu Thr Glu Ala Asn 260 265 270 Leu Lys Gly Glu Ser Ile Trp Lys Leu GluTyr Ile Leu Thr Gln Thr 275 280 285 Tyr Asp Ile Glu Asp Leu Gln Pro GluSer Leu Tyr Gly Leu Ala Lys 290 295 300 Gln Phe Thr Ile Leu Asp Ser LysGln Phe Ile Lys Tyr Tyr Asn Tyr 305 310 315 320 Phe Phe Val Ser Tyr AspSer Ser Val Thr Cys Asp Lys Thr Cys Lys 325 330 335 Ala Phe Gln Ile CysAla Ile Met Asn Leu Asp Asn Ile Ser Tyr Ala 340 345 350 Asp Cys Leu LysGln Leu Tyr Ile Lys His Asn Tyr 355 360 3 1362 DNA Homo sapiens 3atggcgctgg tgcgcgcact cgtctgctgc ctgctgactg cctggcactg ccgctccggc 60ctcgggctgc ccgtggcgcc cgcaggcggc aggaatcctc ctccggcgat aggacagttt 120tggcatgtga ctgacttaca cttagaccct acttaccaca tcacagatga ccacacaaaa 180gtgtgtgctt catctaaagg tgcaaatgcc tccaaccctg gcccttttgg agatgttctg 240tgtgattctc catatcaact tattttgtca gcatttgatt ttattaaaaa ttctggacaa 300gaagcatctt tcatgatatg gacaggggat agcccacctc atgttcctgt acctgaactc 360tcaacagaca ctgttataaa tgtgatcact aatatgacaa ccaccatcca gagtctcttt 420ccaaatctcc aggttttccc tgcgctgggt aatcatgact attggccaca ggatcaactg 480tctgtagtca ccagtaaagt gtacaatgca gtagcaaacc tctggaaacc atggctagat 540gaagaagcta ttagtacttt aaggaaaggt ggtttttatt cacagaaagt tacaactaat 600ccaaacctta ggatcatcag tctaaacaca aacttgtact acggcccaaa tataatgaca 660ctgaacaaga ctgacccagc caaccagttt gaatggctag aaagtacatt gaacaactct 720cagcagaata aggagaaggt gtatatcata gcacatgttc cagtggggta tctgccatct 780tcacagaaca tcacagcaat gagagaatac tataatgaga aattgataga tatttttcaa 840aaatacagtg atgtcattgc aggacaattt tatggacaca ctcacagaga cagcattatg 900gttctttcag ataaaaaagg aagtccagta aattctttgt ttgtggctcc tgctgttaca 960ccagtgaaga gtgttttaga aaaacagacc aacaatcctg gtatcagact gtttcagtat 1020gatcctcgtg attataaatt attggatatg ttgcagtatt acttgaatct gacagaggcg 1080aatctaaagg gagagtccat ctggaagctg gagtatatcc tgacccagac ctacgacatt 1140gaagatttgc agccggaaag tttatatgga ttagctaaac aatttacaat cctagacagt 1200aagcagttta taaaatacta caattacttc tttgtgagtt atgacagcag tgtaacatgt 1260gataagacat gtaaggcctt tcagatttgt gcaattatga atcttgataa tatttcctat 1320gcagattgcc tcaaacagct ttatataaag cacaattact ag 1362 4 1095 DNA Homosapiens 4 atggcgctgg tgcgcgcact cgtctgctgc ctgctgactg cctggcactgccgctccggc 60 ctcgggctgc ccgtggcgcc cgcaggcggc aggaatcctc ctccggcgataggacagttt 120 tggcatgtga ctgacttaca cttagaccct acttaccaca tcacagatgaccacacaaaa 180 gtgtgtgctt catctaaagg tgcaaatgcc tccaaccctg gcccttttggagatgttctg 240 tgtgattctc catatcaact tattttgtca gcatttgatt ttattaaaaattctggacaa 300 gaagcatctt tcatgatatg gacaggggat agcccacctc atgttcctgtacctgaactc 360 tcaacagaca ctgttataaa tgtgatcact aatatgacaa ccaccatccagagtctcttt 420 ccaaatctcc aggttttccc tgcgctgggt aatcatgact attggccacaggtgtatatc 480 atagcacatg ttccagtggg gtatctgcca tcttcacaga acatcacagcaatgagagaa 540 tactataatg agaaattgat agatattttt caaaagtaca gtgatgtcattgcaggacaa 600 ttttatggac acactcacag agacagcatt atggttcttt cagataaaaaaggaagtcca 660 gtaaattctt tgtttgtggc tcctgctgtt acaccagtga agagtgttttagaaaaacag 720 accaacaatc ctggtatcag actgtttcag tatgatcctc gtgattataaattattggat 780 atgttgcagt attacttgaa tctgacagag gcgaatctaa agggagagtccatctggaag 840 ctggagtata tcctgaccca gacctacgac attgaagatt tgcagccggaaagtttatat 900 ggattagcta aacaatttac aatcctagac agtaagcagt ttataaaatactacaattac 960 ttctttgtga gttatgacag cagtgtaaca tgtgataaga catgtaaggcctttcagatt 1020 tgtgcaatta tgaatcttga taatatttcc tatgcagatt gcctcaaacagctttatata 1080 aagcacaatt actag 1095 5 15 PRT Homo sapiens 5 Ser LysGly Ala Asn Ala Ser Asn Pro Gly Pro Phe Gly Asp Val 1 5 10 15

We claim:
 1. A nucleic acid encoding NFIF-14b polypeptide comprising anamino acid sequence as shown in FIG. 1 (SEQ ID NO: 1).
 2. A nucleic acidencoding NFIF-7a polypeptide comprising an amino acid sequence as shownin FIG. 2 (SEQ ID NO: 2).
 3. The nucleic acid of claim 1 wherein saidDNA is a cDNA.
 4. The nucleic acid of claim 2 wherein said DNA is acDNA.
 5. An isolated and purified NFIF-14b polypeptide which inducesNFκB and comprising an amino acid sequence as shown in FIG. 1 (SEQ IDNO: 1).
 6. An isolated and purified NFIF-7a polypeptide which inducesNFκB and comprising an amino acid sequence as shown in FIG. 2 (SEQ IDNO: 2).
 7. A method of increasing expression of NFκB in a patientcomprising introducing into the body of said patient a composition thatinduces NFκB.
 8. The method of claim 7 wherein said compositioncomprises an expression vector comprising a nucleic acid encodingNFIF-14b polypeptide.
 9. The vector of claim 8 selected from the groupconsisting of retroviral vectors, adenoviral vectors, adeno-associatedviral vectors, herpesviral vectors, and naked DNA vectors.
 10. Themethod of claim 7 wherein said composition comprises an expressionvector comprising a nucleic acid encoding NFIF-7a polypeptide.
 11. Thevector of claim 10 selected from the group consisting of retroviralvectors, adenoviral vectors, adeno-associated viral vectors, herpesviralvectors, and naked DNA vectors.
 12. The method of claim 7 wherein saidcomposition comprises a NFIF-14b polypeptide and a pharmaceuticallyacceptable carrier.
 13. The method of claim 7 wherein said compositioncomprises a NFIF-7a polypeptide and a pharmaceutically acceptablecarrier.
 14. A composition for lowering the expression of the NFIF genein a patient comprising an antisense nucleic acid.
 15. A composition forlowering the activity of an NFIF polypeptide in a patient comprising aneutralizing antibody that binds to an NFIF polypeptide and lowers itsactivity.
 16. A composition for lowering the expression of NFIF in apatient comprising a ribozyme that cuts RNA encoding an NFIFpolypeptide.
 17. A method for evaluating whether a test compound iseffective in inhibiting the activity of NFIF-14b based on the expressionof an NFκB-regulated reporter gene comprising: (A) comparing the levelof NFκB-regulated gene expression in a first sample comprising: (1)NFIF-14b; (2) said NFκB-regulated reporter gene; and (3) said testcompound with the level of gene expression in a second sample comprising(4) NFIF-14b; and (5) said NFκB-regulated reporter gene; and (B)determining whether the expression of said reporter gene is lower insaid first sample relative to said second sample.
 18. A method forevaluating whether a test compound is effective in inhibiting theactivity of NFIF-7a based on expression of an NFκB-regulated reportergene comprising: (A) comparing the level of NFκB-regulated geneexpression in a first sample comprising: (1) NFIF-7a; (2) saidNFκB-regulated reporter gene; and (3) said test compound with the levelof gene expression in a second sample comprising: (4) NFIF-7a; and (5)said NFκB-regulated reporter gene; and (B) determining whether theexpression of said reporter gene is lower in said first sample relativeto said second sample.
 19. A method for identifying whether a testcompound can enhance the activity of NFIF-14b based on the expression ofan NFκB-regulated reporter gene comprising: (A) comparing the level ofNFκB-regulated gene expression in a first sample comprising (1)NFIF-14b; (2) said NFκB-regulated reporter gene; and (3) said testcompound with the level of gene expression in a second samplecomprising: (4) NFIF-14b; and (5) said NFκB-regulated reporter gene; and(B) determining whether the expression of said reporter gene is higherin said first sample relative to said second sample.
 20. A method foridentifying compounds which enhance the activity of NFIF-7a based on theexpression of an NFκB-regulated reporter gene comprising: (A) comparingthe level of NFκB-regulated gene expression in a first samplecomprising: (1) NFIF-7a; (2) said NFκB-regulated reporter gene; and (3)said test compound with the level of gene expression in a second samplecomprising: (4) NFIF-7a; and (5) said NFκB-regulated reporter gene; and(B) determining whether the expression of said reporter gene is higherin said first sample relative to said second sample.
 21. A method ofinhibiting expression of NFκB-dependent genes comprising administrationto a patient of a composition that inhibits the activity of NFIF-14b.22. A method of inhibiting expression of NFκB-dependent genes comprisingadministration to a patient of a composition that inhibits the activityof NFIF-7a.
 23. A method of inhibiting inflammation comprisingadministration of a composition that inhibits the activity of NFIF-14b.24. A method of inhibiting inflammation comprising administration of acomposition that inhibits the activity of NFIF-7a.
 25. Use of anisolated NFIF polypeptide comprising an amino acid sequence of FIG. 1(SEQ ID NO: 1) for the manufacture of a medicament intended for thetreatment and/or prevention of an NFκB-regulated inflammatory response.26. Use of a nucleic acid encoding an NFIF polypeptide comprising anamino acid sequence of FIG. 1 (SEQ ID NO: 1) for the manufacture of amedicament intended for the treatment and/or prevention of anNFκB-regulated inflammatory response.
 27. Use of a recombinant vectorcomprising a nucleic acid encoding an NFIF polypeptide comprising anamino acid sequence of FIG. 1 (SEQ ID NO: 1) for the manufacture of amedicament intended for the treatment and/or prevention of anNFκB-regulated inflammatory response.
 28. Use of a defective recombinantviral vector comprising a nucleic acid encoding an NFIF polypeptidecomprising an amino acid sequence of FIG. 1 (SEQ ID NO: 1) for themanufacture of a medicament intended for the treatment and/or preventionof an NFκB-regulated inflammatory response.
 29. Use of an isolated NFIFpolypeptide comprising an amino acid sequence of FIG. 2 (SEQ ID NO: 2)for the manufacture of a medicament intended for the treatment and/orprevention of an NFκB-regulated inflammatory response.
 30. Use of anucleic acid encoding an NFIF polypeptide comprising an amino acidsequence of FIG. 2 (SEQ ID NO: 2) for the manufacture of a medicamentintended for the treatment and/or prevention of an NFκB-regulatedinflammatory response.
 31. Use of a recombinant vector comprising anucleic acid encoding an NFIF polypeptide comprising an amino acidsequence of FIG. 2 (SEQ ID NO: 2) for the manufacture of a medicamentintended for the treatment and/or prevention of an NFκB-regulatedinflammatory response.
 32. Use of a defective recombinant viral vectorcomprising a nucleic acid encoding an NFIF polypeptide comprising anamino acid sequence of FIG. 2 (SEQ ID NO: 2) for the manufacture of amedicament intended for the treatment and/or prevention of anNFκB-regulated inflammatory response.